Soybean plant producing seeds with reduced levels of raffinose saccharides and phytic acid

ABSTRACT

A soybean enzyme, myo-inositol 1-phosphate synthase, whose manipulation results in the alteration of raffinose saccharide, sucrose, phytic acid and inorganic phosphate content of soybean seeds, thus leading to valuable and useful soybean products, has been identifed. Soybean lines with decreased capacity for the synthesis of myo-inositol 1-phosphate in the tissue of developing seeds in comparison to seeds of other soybean lines, have been developed. As taught herein, reduction of myo-inositol 1-phosphate synthase enzymatic activity by any of several means will result in soybean seeds displaying this desirable phenotype.

[0001] This application is a continuation-in-part of InternationalApplication No. PCT/US98/06822 filed Apr. 7, 1998, which application wasa continuation-in-part of U.S. application Ser. No. 08/835,751 filedApr. 8, 1997.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology andgenetics. More specifically, this invention pertains to soybean plantshaving in their seeds significantly lower contents of raffinose,stachyose and phytic acid and significantly higher contents of sucroseand inorganic phosphate. This phenotype is the result of a heritable,decreased capacity for the production of myo-inositol 1-phosphate.

BACKGROUND OF THE INVENTION

[0003] Raffinose saccharides are a group of D-galactose-containingoligosaccharides of sucrose that are widely distributed in plants.Raffinose saccharides are characterized by having the general formulaO-α-D-galactopyranosyl-(1→6)_(n)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranosidewhere n=1 through n=4 are known respectively as raffinose, stachyose,verbascose, and ajugose. In soybean seeds, raffinose and stachyose arethe raffinose saccharides that are present in greatest quantity. Incontrast, verbascose and ajugose are minor components and are generallynot detected by standard analytical methods.

[0004] Extensive botanical surveys of the occurrence of raffinosesaccharides have been reported in the scientific literature [Dey, P. M.In Biochemistry of Storage Carbohydrates in Green Plants, AcademicPress, London, (1985) pp 53-129]. Raffinose saccharides are thought tobe second only to sucrose among the nonstructural carbohydrates withrespect to abundance in the plant kingdom. In fact, raffinosesaccharides may be ubiquitous, at least among higher plants. Raffinosesaccharides accumulate in significant quantities in the edible portionof many economically significant crop species. Examples include soybean(Glycine max L. Merrill), sugar beet (Beta vulgaris), cotton (Gossylpiumhirsutum L.), canola (Brassica sp.) and all of the major edibleleguminous crops including beans (Phaseolus sp.), chick pea (Cicerarietinum), cowpea (Vigna unguiculata), mung bean (Vigna radiata), peas(Pisum sativum), lentil (Lens culinaris) and lupine (Lupinus sp.).

[0005] Although abundant in many species, raffinose saccharides are anobstacle to the efficient utilization of some economically importantcrop species. Raffinose saccharides are not digested directly byanimals, primarily because α-galactosidase is not present in theintestinal mucosa [Gitzelmann and Auricchio.(1965) Pediatrics36:231-236; Rutloffet al. (1967) Nahrung. 11:39-46]. However, microflorain the lower gut are readily able to ferment the raffinose saccharideswhich results in an acidification of the gut and production of carbondioxide, methane and hydrogen [Murphy et al. (1972) J. Agr. Food Chem.20:813-817; Cristofaro et al. In Sugars in Niitrition, (1974) Chapter20, 313-335; Reddy et al. (1980) J. Food Science 45:1161-1164]. Theresulting flatulence can severely limit the use of leguminous plants inanimal, including human, diets. It is unfortunate that the presence ofraffinose saccharides restricts the use of soybeans in animal, includinghuman, diets because otherwise this species is an excellent source ofprotein and fiber.

[0006] The soybean is well-adapted to machinery and facilities forharvesting, storing and processing that are widely available in manyparts of the world. In the U.S. alone, approximately 28 million metrictons of meal were produced in 1988 [Oil Crops Situation and OutlookReport, April 1989, U.S. Dept. of Agriculture, Economic ResearchService]. Typically, hulls are removed and then the oil is extractedwith hexane in one of several extraction systems. The remaining defattedflakes can then be used for a variety of commercial soy protein products[Soy Protein Products, Characteristics, Nutritional Aspects andUtilization (1987) Soy Protein Council]. Foremost among these in volumeof use is soybean meal, the principle source of protein in diets usedfor animal feed, especially those for monogastric animals such aspoultry and swine.

[0007] Although the soybean is an excellent source of vegetable protein,there are inefficiencies associated with its use that appear to be dueto the presence of raffinose saccharides. Compared to maize, the otherprimary ingredient in animal diets, gross energy utilization for soybeanmeal is low [Potter and Potchahakorn. In: Proceedings World SoybeanConference III, (1984) 218-224]. For example, although soybean mealcontains approximately 6% more gross energy than ground yellow corn, ithas about 40 to 50% less metabolizable energy when fed to chickens. Thisinefficiency of gross energy utilization does not appear to be due toproblems in digestion of the protein fraction of the meal, but ratherdue to the poor digestion of the carbohydrate portion of the meal. Ithas been reported that removal of raffinose saccharides from soybeanmeal by ethanol extraction results in a large increase in themetabolizable energy for broilers [Coon, C. N. et al. In: ProceedingsSoybean Utilization Alternatives, University of Minnesota, (1988)203-211]. Removal of the raffinose saccharides was associated withincreased utilization of the cellulosic and hemicellulosic fractions ofthe soybean meal.

[0008] A variety of processed vegetable protein products are producedfrom soybean. These range from minimally processed, defatted items suchas soybean meal, grits, and flours to more highly processed items suchas soy protein concentrates and soy protein isolates. In other soyprotein products the oil is not extracted, full-fat soy flour forexample. In addition to these processed products, there are also anumber of speciality products based on traditional Oriental processes,which utilize the entire bean as the starting material. Examples includesoy milk, soy sauce, tofu, natto, miso, tempeh and yuba.

[0009] Examples of use of soy protein products in human foods includesoy protein concentrates, soy protein isolates, textured soy protein,soy milk and infant formula. Facilities and methods to produce proteinconcentrates and isolates from soybeans are available across the world.Soy protein concentrates and soy protein isolates are used primarily asfood and feed ingredients. Conditions typically used to prepare soyprotein isolates have been described by [Cho, et al, (1981) U.S. Pat.No. 4,278,597; Goodnight, et al, (1978) U.S. Pat. No. 4,072,670]. Soyprotein concentrates are produced by three basic processes: acidleaching (at about pH 4.5), extraction with alcohol (about 55-80%), anddenaturing the protein with moist heat prior to extraction with water.Conditions typically used to prepare soy protein concentrates have beendescribed by Pass [(1975) U.S. Pat. No. 3,897,574; Campbell et al.,(1985) in New Protein Foods, ed. by Altschul and Wilcke, Academic Press,Vol. 5, Chapter 10, Seed Storage Proteins, pp 302-338.] One of theproblems faced by producers of soy protein concentrates and isolates isthe challenge of selectively purifying the protein away from theraffinose saccharides. Considerable equipment and operating costs areincurred as a result of removing the large amounts of raffinosesaccharides that are present in soybeans.

[0010] The problems and costs associated with raffinose saccharidescould be reduced or eliminated through the availability of genes thatconfer a reduction of raffinose saecharide content of soybean seeds.Such genes could be used to develop soybean varieties having inherentlyreduced raffinose saccharide content. Soybean varieties with inherentlyreduced raffinose saccharide content would improve the nutritionalquality of derived soy protein products and reduce processing costsassociated with the removal of raffinose saccharides. Low raffinosesaccharide soybean varieties would be more valuable than conventionalvarieties for animal and human diets and would allow mankind to morefully utilize the desirable nutritional qualities of this edible legume.

[0011] U.S. Pat. No. 5,710,365 discloses soybeans that are low in totalraffinose saccharides, and describes the advantages and preparation ofsoy protein products derived from those soybeans. However, U.S. Pat. No.5,710,365 does not report or suggest soybeans possessing high levels offree phosphate and sucrose, low levels of phytate, and which are alsolow in raffinose saccharides.

[0012] myo-Inositol hexaphosphate (also know as “phytic acid”) andraffinose saccharides share myo-inositol as a common intermediate intheir synthesis [Ishitani, M. et al., (1996) The Plant Journal9:537-548]. Like raffinose saccharides, phytic acid is a nearlyubiquitous component of angiosperm seeds [Raboy, V. In: InositolMetaholism in Plants (1990) Wiley-Liss, New York, pp 55-76]. Whilephytic acid typically accounts for 50 to 70% of the total phosphate inseeds such as soybean and corn, that phosphate is only poorly availableto mono-gastric animals. In addition to being only partially digestible,the presence of phytic acid in animal rations leads to excretion ofother limiting nutrients such as essential amino acids, calcium and zinc[Mroz, Z. et al., (1994) J. Animal Sci. 72.126-132; Fox et al., In:Nutritional Toxicology Vol. 3, Academic Press, San Diego (1989) pp.59-96]. Since soybean meal is a major portion of many animal feedrations, a meal with decreased amounts of phytic acid along withincreased amounts of available phosphate should lead to improved feedefficiency in soy containing rations. Indeed enzymatic treatment ofsoybean meal containing rations to partially hydrolyze the phosphategroups from phytic acid improves both phosphate availability and theavailability of other limiting nutrients [Mroz et al., supra; Pen etal., (1993) Biotechnology 11:811-814].

[0013] Surveys of commercial and wild soybean germplasm indicate thatlimited genetic variability for seed phytic acid content exists [Raboyet al., (1984) Crop Science 24:431-434]. In light of these factors, itis apparent that soybean plants with heritable, substantially reducedlevels of raffinose saccharides and phytic acid in their seeds areneeded.

SUMMARY OF THE INVENTION

[0014] The instant invention pertains to a soybean plant with aheritable phenotype of (i) a seed phytic acid content of less than 17μmol/g, (ii) a seed content of raffinose plus stachyose of less than14.5 μmol/g, and (iii) a seed sucrose content of greater than 200μmol/g, the phenotype due to a decreased capacity for the synthesis ofmyo-inositol 1-phosphate in the seeds of the plant. The invention alsopertains to seeds derived from this plant.

[0015] In addition, this invention pertains to an isolated nucleic acidfragment encoding a soybean myo-inositol 1-phosphate synthase or itscomplement, and a chimeric gene comprising this nucleic acid fragment ora subfragment of this nucleic acid fragment, operably linked to suitableregulatory sequences, wherein expression of the chimeric gene results ina decrease in expression of a native gene encoding myo-inositol1-phosphate synthase.

[0016] Another embodiment of the instant invention is an isolatednucleic acid fragment encoding a mutant myo-inositol 1-phosphatesynthase, the mutant enzyme having decreased capacity for the synthesisof myo-inositol 1-phospate.

[0017] Yet another embodiment of the instant invention is a soybeanplant having in its genome a chimeric gene comprising a nucleic acidfragment encoding a soybean myo-inositol 1-phosphate synthase or thecomplement of the nucleic acid fragment, operably linked to suitableregulatory sequences, wherein expression of the chimeric gene results ina decrease in expression of a native gene encoding a soybeanmyo-inositol-1-phosphate synthase.

[0018] Still another embodiment of the instant invention is a soybeanplant homozygous for at least one gene encoding a mutant myo-inositol1-phosphate synthase having decreased capacity for the synthesis ofmyo-inositol 1-phosphate, the gene conferring a heritable phenotype of(i) a seed phytic acid content of less than 17 μmol/g, (ii) a seedcontent of raffinose plus stachyose of less than 14.5 μmol/g, and (iii)a seed sucrose content of greater than 200 μmol/g.

[0019] The instant invention also pertains to seeds and protein productsderived from the seeds of any of the plants described above.

[0020] Yet another embodiment of the instant invention is a method formaking a soybean plant with a heritable phenotype of (i) a seed phyticacid content less than 17 μmol/g, (ii) a seed content of raffinose plusstachyose of less than 14.5 μmol/g, and (iii) a seed sucrose content ofgreater than 200 μmol/g.

[0021] The instant invention also embodies a method for producing a soyprotein product derived from the seeds of soybean plant that have a seedphytic acid content less than 17 μmol/g, (ii) a seed content ofraffinose plus stachyose of less than 14.5 μmol/g, and (iii) a seedsucrose content of greater than 200 μmol/g.

[0022] Finally, the instant invention embodies a method of using asoybean plant homozygous for at least one gene encoding a mutantmyo-inositol 1-phosphate synthase having decreased capacity for thesynthesis of myo-inositol 1-phosphate, the gene conferring a heritablephenotype of (i) a seed phytic acid content less than 17 μmol/g, (ii) aseed content of raffinose plus stachyose of less than 14.5 μmol/g, and(iii) a seed sucrose content of greater than 200 μmol/g to produceprogeny lines, the method comprising (a) crossing a soybean plantcomprising a mutant myo-inositol 1-phosphate synthase having decreasedcapacity for the synthesis of myo-inositol 1-phosphate with any soybeanparent which does not comprise the mutation, to yield a F1 hybrid; (b)selfing the F1 hybrid for at least one generation; and (c) identifyingthe progeny of step (b) homozygous for at least one gene encoding amutant myo-inositol 1-phosphate synthase having decreased capacity forthe synthesis of myo-inositol 1-phosphate, the gene conferring aheritable phenotype of (i) a seed phytic acid content less than 17μmol/g, (ii) a seed content of raffinose plus stachyose of less than14.5 μmol/g, and (iii) a seed sucrose content of greater than 200μmol/g.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

[0023] The invention can be more fully understood from the figures andthe Sequence Listing which form a part of this application.

[0024]FIG. 1 is a diagram of glucose metabolism to phytic acid,raffinose and stachyose in soybean seeds. The common co-factors, ATP,ADP, UTP, UDP, pyrophosphate and inorganic phosphate (Pi) are not shown.The enzymes are listed by abbreviation: hexokinase (HK);phosphoglucoisomerase (PGI); UDPglucose pyrophosphorylase (UDPGPP);UDPglucose 4′ epimerase (UDPG 4′E); myo-inositol 1-phosphate synthase(MI 1-PS); myo-inositol 1-phosphatase (MI 1-Pase); myo-inositol1-phosphate kinase (MI 1-PK); galactinol synthase (GAS); sucrosesynthase (SucS); raffinose synthase (RS); and stachyose synthase (SS).

[0025]FIG. 2 is an alignment of the nucleotide sequences encodingmyo-inositol 1-phosphate synthases from obtained from the wild typesoybean cultivar Wye (SEQ ID NO: 1 and SEQ ID NO: 15) and the nucleotidesequences encoding myo-inositol 1-phosphate synthases obtained from themutant soybean lines LR33, 29004JP01, 29010CP01 and 29018JP03 thatdemonstrate the low phytate, low raffinose saccharide, high sucrosephenotype described herein (SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 11and SEQ ID NO: 13, respectively). Bold, italicized residues indicatepositions of nucleotide sequence variability between the sequencesencoding the two myo-inositol 1-phosphate synthase isozymes. Bold,italicized and underlined residues indicate nucleotide changes thatencode amino acid substitutions between the mutant myo-inositol1-phosphate synthase and the corresponding wild type enzyme.

[0026]FIG. 3 is an alignment of the amino acid sequences of themyo-inositol 1-phosphate synthases from obtained from the wild typesoybean cultivar Wye (SEQ ID NO: 2 and SEQ ID NO: 16) and the amino acidsequences of myo-inositol 1-phosphate synthases obtained from the mutantsoybean lines LR33, 29004JP01, 29010CP01 and 29018JP03 that demonstratethe low phytate, low raffinose saccharide, high sucrose phenotypedescribed herein (SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ IDNO: 14, respectively). Bold, italicized residues indicate positions ofamino acid sequence variability between the sequences encoding the twomyo-inositol 1-phosphate synthase isozymes. Bold, italicized andunderlined residues indicate amino acid changes that encode amino acidsubstitutions between the mutant myo-inositol 1-phosphate synthase andthe corresponding wild type enzyme.

[0027] The Sequence Listing contains the one letter codes for nucleotidesequence characters and the three letter codes for amino acids asdefined in the IUPAC-IUB standards described in Nucleic Acids Research13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373(1984). The symbols and format used for all nucleotide and amino acidsequence data comply with the rules governing nucleotide and/or aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §1.821-1.825.

[0028] SEQ ID NO: 1 is the 5′ to 3′ nucleotide sequence of the 1782bases of the cDNA encoding the wild type soybean myo-inositol1-phosphate synthase present in clone p5bmi-1ps.

[0029] SEQ ID NO: 2 is the 510 amino acid sequence deduced from the openreading frame in SEQ ID NO: 1.

[0030] SEQ ID NO: 3 is the nucleotide sequence of the upstream (5′)primer used in the isolation of the myo-inositol 1-phosphate synthasecDNA from soybean line LR33.

[0031] SEQ ID NO: 4 is the nucleotide sequence of the downstream (3′)primer used in the isolation of the myo-inositol 1-phosphate synthasecDNA from soybean line LR33.

[0032] SEQ ID NO: 5 is the nucleotide sequence of the 1533 bases of cDNAencoding the LR33 myo-inositol 1-phosphate synthase present in cloneLR33-10.

[0033] SEQ ID NO: 6 is the 510 amino acid sequence deduced from the openreading frame in SEQ ID NO: 5.

[0034] SEQ ID NO: 7 is the nucleotide sequence of the upstream (5′)primer used for PCR amplification of the wild type allele encodingmyo-inositol 1-phosphate synthase from genomic DNA samples.

[0035] SEQ ID NO: 8 is the nucleotide sequence of the upstream (5′)primer used for PCR amplification of the LR33 allele encodingm)o-inositol 1-phosphate synthase from genomic DNA samples.

[0036] SEQ ID NO: 9 is the nucleotide sequence for myo-inositol1-phosphate synthase cDNA obtained from mutant line 29004JP01.

[0037] SEQ ID NO: 10 is the 510 amino accid sequence deduced from thecDNA sequence set forth in SEQ ID NO: 9.

[0038] SEQ ID NO: 11 is the nucleotide sequence for myo-inositol1-phosphate synthase cDNA obtained from mutant line 29010CP01.

[0039] SEQ ID NO: 12 is the 510 amino accid sequence deduced from thecDNA sequence set forth in SEQ ID NO: 11.

[0040] SEQ ID NO: 13 is the nucleotide sequence for myo-inositol1-phosphate synthase cDNA obtained from mutant line 29018JP03.

[0041] SEQ ID NO: 14 is the 510 amino accid sequence deduced from thecDNA sequence set forth in SEQ ID NO: 13.

[0042] SEQ ID NO: 15 is the nucleotide sequence obtained from a cDNA fora second wild type form of myo-inositol 1-phosphate synthase.

[0043] SEQ ID NO: 16 is the 510 amino accid sequence deduced from thecDNA sequence set forth in SEQ ID NO: 15.

Biological Deposits

[0044] The following biological materials have been deposited under theterms of the Budapest Treaty at American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209, and bear thefollowing accession numbers: Designation Material Accession Number Dateof Deposit LR33 Seed ATCC 97988 Apr. 17, 1997 4E76 Seed ATCC 97971 Apr.4, 1997 p5bmi-1ps Plasmid ATCC 97970 Apr. 4, 1997 29004JP01 Seed ATCCXXXXX 29010CP01 Seed ATCC YYYYY 29018JP03 Seed ATCC ZZZZZ

DETAILED DESCRIPTION

[0045] Soybean lines that are lower in phytic acid and raffinosesaccharide content and higher in sucrose and inorganic phosphateconcentration than wild type soybean lines are described. These lineswere developed by selection after chemical mutagenesis. The biochemicalbasis for this desirable phenotype has been characterized as a geneticdefect leading to lower myo-inositol 1-phosphate synthase activity inthe mutant lines compared to their wild type counterparts. Moreover, ithas been shown that the mutant phenotype may be displayed by soybeanlines that carry a mutant form of the enzyme myo-inositol 1-phosphatesynthase, or in lines that appear to possess a wild type form of theenzyme but are otherwise deficient in production of this enzyme. Bothtypes of genetic defects are shown to be allelic. Accordingly, theinstant specification enables one skilled in this art to create soybeanlines with the instant low phytate, low raffinose saccharide, highsucrose, high inorganic phosphate phenotype by decreasing or eliminatingmyo-inositol 1-phosphate synthase activity. As described herein,decreased myo-inositol 1-phosphate synthase activity may be achieved byany of several methods known to one skilled in the art such as chemicalmutagenesis and selection, traditional plant breeding using a mutantthat is deficient in myo-inositol 1-phosphate synthase activity as abreeding parent, or by techniques of gene silencing such as antisenseinhibition of gene expression or co-suppression.

[0046] In the context of this disclosure, a number of terms shall beutilized. As used herein, “soybean” refers to the species Glycine max,Glycine soja, or any species that is sexually cross compatible withGlycine max. A “line” is a group of plants of similar parentage thatdisplay little or no genetic variation between individuals for a leastone trait. Such lines may be created by one or more generations ofself-pollination and selection, or vegetative propagation from a singleparent including by tissue or cell culture techniques. “Mutation” refersto a detectable and heritable genetic change (either spontaneous orinduced) not caused by segregation or genetic recombination. “Mutant”refers to an individual, or lineage of individuals, possessing amutation. A “population” is any group of individuals that share a commongene pool. In the instant invention, this includes M1, M2, M3, M4, F1,and F2 populations. As used herein, an “M1 population” is the progeny ofseeds (and resultant plants) that have been exposed to a mutagenicagent, while “M2 population” is the progeny of self-pollinated M1plants, “M3 population” is the progeny of self-pollinated M2 plants, and“M4 population” is the progeny of self-pollinated M3 plants. As usedherein, an “F1 population” is the progeny resulting from crosspollinating one line with another line. The format used herein to depictsuch a cross pollination is “female parent*male parent”. An “F2population” is the progeny of the self-pollinated F1 plants. An“F2-derived line” or “F2 line” is a line resulting from theself-pollination of an individual F2 plant. An F2-derived line can bepropagated through subsequent generations (F3, F4, F5 etc.) by repeatedself-pollination and bulking of seed from plants of said F2-derivedline.

[0047] The term “nucleic acid” refers to a large molecule which can besingle-stranded or double-stranded, composed of monomers (nucleotides)containing a sugar, a phosphate and either a purine or pyrimidine. A“nucleic acid fragment” is a fraction of a given nucleic acid molecule.A “subfragment” refers to a contiguous portion of a nucleic acidfragment comprising less than the entire nucleic acid fragment.“Complementary” refers to the specific pairing of purine and pyrimidinebases that comprise nucleic acids: adenine pairs with thymine andguanine pairs with cytosine. Thus, the “complement” of a first nucleicacid fragment refers to a second nucleic acid fragment whose sequence ofnucleotides is complementary to the first nucleic acid sequence.

[0048] In higher plants, deoxyribonucleic acid (DNA) is the geneticmaterial while ribonucleic acid (RNA) is involved in the transfer of theinformation in DNA into proteins. A “genome” is the entire body ofgenetic material contained in each cell of an organism. The term“nucleotide sequence” refers to the sequence of DNA or RNA polymers,which can be single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases capable ofincorporation into DNA or RNA polymers. The term “oligomer” refers toshort nucleotide sequences, usually up to 100 bases long. As usedherein, the term “homologous to” refers to the relatedness between thenucleotide sequence of two nucleic acid molecules or between the aminoacid sequences of two protein molecules. Estimates of such homology areprovided by either DNA-DNA or DNA-RNA hybridization under conditions ofstringency as is well understood by those skilled in the art (Hames andHiggins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.); or by the comparison of sequence similarity between two nucleicacids or proteins, such as by the method of Needleman et al. (J. Mol.Biol. (1970) 48:443-453).

[0049] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the polypeptide or protein encoded by thenucleotide sequence. “Substantially similar” also refers to nucleic acidfragments wherein changes in one or more nucleotide bases does notaffect the ability of the nucleic acid fragment to mediate alteration ofgene expression by gene silencing through for example antisense orco-suppression technology. “Substantially similar” also refers tomodifications of the nucleic acid fragments of the instant inventionsuch as deletion or insertion of one or more nucleotides that do notsubstantially affect the functional properties of the resultingtranscript vis-à-vis the ability to mediate gene silencing or alterationof the functional properties of the resulting protein molecule. It istherefore understood that the invention encompasses more than thespecific exemplary nucleotide or amino acid sequences.

[0050] For example, it is well known in the art that antisensesuppression and co-suppression of gene expression may be accomplishedusing nucleic acid fragments representing less than the entire codingregion of a gene, and by nucleic acid fragments that do not share 100%sequence identity with the gene to be suppressed. Moreover, alterationsin a nucleic acid fragment which result in the production of achemically equivalent amino acid at a given site, but do not effect thefunctional properties of the encoded polypeptide or protein, are wellknown in the art. Thus, a codon for the amino acid alanine, ahydrophobic amino acid, may be substituted by a codon encoding anotherless hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, changeswhich result in substitution of one negatively charged residue foranother, such as aspartic acid for glutamic acid, or one positivelycharged residue for another, such as lysine for arginine, can also beexpected to produce a functionally equivalent product. Nucleotidechanges which result in alteration of the N-terminal and C-terminalportions of the polypeptide or protein molecule would also not beexpected to alter the activity of the polypeptide or protein. Each ofthe proposed modifications is well within the routine skill in the art,as is determination of retention of biological activity of the encodedproducts.

[0051] Moreover, substantially similar nucleic acid fragments may alsobe characterized by their ability to hybridize, under stringentconditions (0.1×SSC, 0.1% SDS, 65° C.), with the nucleic acid fragmentsdisclosed herein.

[0052] Substantially similar nucleic acid fragments of the instantinvention may also be characterized by the percent similarity of theirnucleotide sequences to the nucleotide sequences of the nucleic acidfragments disclosed herein, as determined by algorithms commonlyemployed by those skilled in this art. Preferred are those nucleic acidfragments whose nucleotide sequences are 80% similar to the nucleotidesequences reported herein. More preferred nucleic acid fragments whosenucleotide sequences are 90% similar to the nucleotide sequencesreported herein. Most preferred are nucleic acid fragments whosenucleotide sequences are 95% similar to the nucleotide sequencesreported herein. Sequence alignments and percent similarity calculationswere performed using the Megalign program of the LASARGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences was performed using the Clustal method ofalignment (Higgins, D. G. and Sharp, P. M. (1989) CABIOS. 5:151-153)with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Default parameters for pairwise alignments using the Clustal method wereKTUPLE 2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4.

[0053] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding) and following (3′ non-coding) the coding region. “Native”gene refers to an isolated gene with its own regulatory sequences asfound in nature. “Chimeric gene” refers to a gene that comprisesheterogeneous regulatory and coding sequences not found in nature.“Endogenous” gene refers to the native gene normally found in itsnatural location in the genome and is not isolated. A “foreign” generefers to a gene not normally found in the host organism but that isintroduced by gene transfer. “Pseudo-gene” refers to a genomicnucleotide sequence that does not encode a functional enzyme.

[0054] “Coding sequence” refers to a DNA sequence that codes for aspecific protein and excludes the non-coding sequences. It mayconstitute an “uninterrupted coding sequence”, i.e., lacking an intronor it may include one or more introns bounded by appropriate splicejunctions. An “intron” is a nucleotide sequence that is transcribed inthe primary transcript but that is removed through cleavage andre-ligation of the RNA within the cell to create the mature mRNA thatcan be translated into a protein.

[0055] “Initiation codon” and “termination codon” refer to a unit ofthree adjacent nucleotides in a coding sequence that specifiesinitiation and chain termination, respectively, of protein synthesis(mRNA translation). “Open reading frame” refers to the coding sequenceuninterrupted by introns between initiation and termination codons thatencodes an amino acid sequence.

[0056] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect copy of the DNA sequence, it is referred to asthe primary transcript or it may be a RNA sequence derived fromposttranscriptional processing of the primary transcript and is thenreferred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNAthat is without introns and that derived from mRNA. “Sense” RNA refersto an RNA transcript that includes the mRNA. “Antisense RNA” refers toan RNA transcript that is complementary to all or part of a targetprimary transcript or mRNA and that blocks the expression of a targetgene by interfering with the processing, transport and/or translation ofits primary transcript or mRNA. The complementarity of an antisense RNAmay be with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence. In addition, as used herein, antisense RNA may contain regionsof ribozyme sequences that increase the efficacy of antisense RNA toblock gene expression. “Ribozyme” refers to a catalytic RNA and includessequence-specific endoribonucleases.

[0057] As used herein, “suitable regulatory sequences” refer tonucleotide sequences in native or chimeric genes that are locatedupstream (5′), within, and/or downstream (3′) to the nucleic acidfragments of the invention, which control the expression of the nucleicacid fragments of the invention.

[0058] “Promoter” refers to a DNA sequence in a gene, usually upstream(5′) to its coding sequence, which controls the expression of the codingsequence by providing the recognition for RNA polymerase and otherfactors required for proper transcription. In artificial DNA constructspromoters can also be used to transcribe antisense RNA. Promoters mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions. It may alsocontain enhancer elements. An “enhancer” is a DNA sequence which canstimulate promoter activity. It may be an innate element of the promoteror a heterologous element inserted to enhance the level and/ortissue-specificity of a promoter. “Constitutive promoters” refers tothose that direct gene expression in all tissues and at all times.“Tissue-specific” or “development-specific” promoters as referred toherein are those that direct gene expression almost exclusively inspecific tissues, such as leaves or seeds, or at specific developmentstages in a tissue, such as in early or late embryogenesis,respectively. The term “expression”, as used herein, refers to thetranscription and stable accumulation of the sense (mRNA) or theantisense RNA derived from the nucleic acid fragment(s) of the inventionthat, in conjunction with the protein apparatus of the cell, results inaltered levels of myo-inositol 1-phosphate synthase. “Antisenseinhibition” refers to the production of antisense RNA transcriptscapable of preventing the expression of the target protein.“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in normal ornon-transformed organisms. “Cosuppression” refers to the expression of aforeign gene which has substantial homology to an endogenous generesulting in the suppression of expression of both the foreign and theendogenous gene. “Altered levels” refers to the production of geneproduct(s) in transgenic organisms in amounts or proportions that differfrom that of normal or non-transformed organisms. The present inventionalso relates to vectors which include nucleotide sequences of thepresent invention, host cells which are genetically engineered withvectors of the invention and the production of polypeptides of theinvention by recombinant techniques. “Transformation” herein refers tothe transfer of a foreign gene into the genome of a host organism andits genetically stable inheritance. “Fertile” refers to plants that areable to propagate sexually.

[0059] “Raffinose saccharides” refers to the family of oligosaccharideswith the general formulaO-α-D-galactopyranosyl-(1→6)_(n)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranosidewhere n=1 to 4. In soybean seeds, the term refers more specifically tothe members of the family containing one (raffinose) and two (stachyose)galactose residues. Although higher galactose polymers are known (e.g.,verbascose and ajugose), the content of these higher polymers in soybeanis below standard methods of detection and therefore usually do notcontribute significantly to total raffinose saccharide content. As usedherein, “raffinose plus stachyose” refers to the sum of theconcentration of raffinose plus the concentration of stachyose.

[0060] “Soy protein product” refers to products prepared from processingof soybeans that contain a significant fraction of their dry weight asprotein. Soy protein products include but are not limited to soy proteinconcentrates, soy protein isolates, textured soy protein, soy milk,soybean meal, soy grits, and soy flours.

[0061] “Plants” refer to photosynthetic organisms, both eukaryotic andprokaryotic, whereas the term “Higher plants” refers to eukaryoticplants.

[0062] This invention provides a mutated form of a soybean gene andmethods to improve the carbohydrate and phytic acid composition ofsoybean seeds and derived products. The invention teaches examples of amethod to identify mutations in this gene and to use derived mutantsoybean lines to reduce the raffinose saccharide and phytic acid contentof soybean seeds. This invention also teaches methods of using genesilencing technology and the soybean gene sequence formyo-inositol-1-phosphate synthase discovered in the instant invention toreduce the raffinose saccharide content in soybean seeds.

[0063] Seeds derived from the plants of the present invention express animproved soluble carbohydrate content relative to commercial varieties.The improvements result in a reduced total raffinose plus stachyosecontent. The carbohydrate profile of these lines is dramaticallydifferent from the profiles seen in elite or germplasm lines used in orproduced by other soybean breeding programs.

[0064] Two separate methods to produce the novel soybean genes of thepresent invention are taught. The first approach marks the firstsuccessful attempt to induce a mutation conferring low raffinose plusstachyose content. This approach resulted in the discovery of two majorgenes, one of which is described in detail in this invention, that canbe used to develop soybean lines that are superior (in terms of reducingcombined raffinose and stachyose content) to any lines previouslyreported. The second approach utilizes the gene sequences taught in thisinvention applied in transgenic methods of specific gene silencing toachieve results similar to those obtained through random mutagenesis andscreening.

[0065] Applicants initially sought to introduce random mutations in thegenome of wild-type soybeans by chemical mutagenesis. The instantinvention employed NMU (N-nitroso-N-methylurea) as the mutagenic agent,although other agents known to alter DNA structure and sequence couldhave been used. Following treatment NMU, soybean seeds were sown forseveral generation and screened for the desired phenotype; of primaryimportance was the alteration of raffinose saccharide content. Initialscreening of mutagenized soybean populations revealed two lines, LR33and LR28, that appeared to be low in raffinose saccharides (LR28 isdisclosed in World Patent Publication WO93/07742). The low raffinosesaccharide phenotype of LR33 was demonstrated to be inheritable byanalysis of three subsequent generations of LR33 produced byself-fertilization (Table 1).

[0066] The physiological defect in LR33 leading to the unique phenotypedisplayed by this line was identified and characterized by conducting aseries of elegant genetic and biochemical studies (see Example 2,infra). The defect in LR33 was shown to be genetically and biochemicallydistinct from the mutation in LR28 that leads to the low stachyosephenotype of that line. Moreover, the mutation in LR33 demonstratesgreater pleiotrophy than the defect in LR28; the instant specificationdemonstrates that the LR33 phenotype includes not only reduced raffinosesaccharide content, but also results in alterations in seed phytic acid,inorganic phosphate and sucrose levels. Further analyses confirmed thatgenetic information derived from LR33 alone could confer this uniquephenotype on progeny soybean lines, and that the mutant gene or genes inLR33 are not simply genetic modifiers that enhance the phenotypicexpression of genes derived from other mutant soybean lines.

[0067] The specific biochemical defect responsible for the heritablephenotype demonstrated by LR33 and its progeny has been identified. Thiswas accomplished by consideration of the biosynthesis of raffinosesaccharides and the control of phytic acid and inorganic phosphatelevels in soybean seeds. Based upon these known biosynthetic pathways, aseries of biochemical studies and subsequent molecular genetic analysesidentified defect in LR33 seeds as an alteration in myo-inositol1-phosphate synthase activity, leading to a decreased capacity forsynthesis of myo-inositol 1-phosphate.

[0068] The biosynthesis of raffinose and stachyose has been fairly wellcharacterized [see Dey, P. M. In Biochemistry of Storage Carbohydratesin Green Plants (1985)]. Myo-Inositol hexaphosphate or phytic acid andraffinose saccharides share myo-inositol as a common intermediate intheir synthesis [Ishitani,M et al. The Plant Journal (1996) 9:537-548].Starting with glucose as a carbon source, the pathway describing thesynthesis of phytic acid, raffinose and stachyose in maturing soybeanseeds is shown in FIG. 1.

[0069] By the interconversions shown in FIG. 1, either glucose orsucrose can be the starting material for the polyol portion of phyticacid, all of the hexoses that make up raffinose and stachyose and there-cycled portion of the galactose donor to raffinose synthase andstacyhose synthase, myo-inositol. The end products of theseinterconversions that accumulate in mature, wild type soybean seeds are,in order of prominance by mass, sucrose, stachyose, phytic acid, andraffinose.

[0070] The committed reaction of raffinose saccharide biosynthesisinvolves the synthesis of galactinol(O-α-D-galactopyranosyl-(1→1)-myo-inositol) from UDPgalactose andmyo-inositol. The enzyme that catalyzes this reaction is galactinolsynthase. Synthesis of raffinose and higher homologues in the raffinosesaccharide family from sucrose is catalyzed by thegalactosyltransferases raffinose synthase and stachyose synthase.

[0071] Control over the ratio of these end products may be affected byaltering the rate of conversion at many of the enzyme catalyzed steps inFIG. 1. That control can be affected by altering enzyme expression levelor by altering the intrinsic activity of the enzyme. The resulting mixof end products coming from the modified pathway may then comprise newproportions of the original end products as well as new product mixeswhich include accumulations of some of the normal intermediates. Theexact mix and composition will depend upon both the enzyme which hasbeen altered in its activity and the degree of that alteration.

[0072] The six enzymes myo-inositol 1-phosphate synthase, myo-inositol1-phosphatase, UDP-glucose 4′ epimerase, galactinol synthase, raffinosesynthase, and stachyose synthase could be reduced in activity todecrease either raffinose or stachyose synthesis without decreasingsucrose content. Of these six, the three enzymes unique to raffinose andstachose synthesis could be decreased in activity without decreasingphytic acid content. Only myo-inositol 1-phosphate synthase appears tobe involved in the synthesis of all three end products and may thereforechange the amount of all three end products simultaneosly if itsactivity is decreased.

[0073] The instant invention teaches the ability to simultaneouslyreduce raffinose saccharide and phytic acid content and increase sucroseand inorganic phosphate content in soybean seeds by reducingmyo-inositol 1-phosphate synthase activity in the cells of soybeanseeds. The instant examples describe generation and discovery of amutant form of this enzyme wherein a point mutation in the nucleotidesequence encoding this enzyme results in an amino acid substitutionwhich, in turn, lowers intracellular enzymatic activity. It is wellknown to the skilled artisan that other mutations within the codingregion for myo-inositol 1-phosphate synthase can result in decreasedenzymatic activity and thus result in the instant seed phenotype. Usingwell known techniques of heterologous gene expression and in vitromutagenesis, and employing the various enzymatic assays describedherein, the skilled artisan could identify other mutations within themyo-inositol 1-phosphate synthase coding region that result in decreasedenzymatic activity without undue experimentation. These mutatedmyo-inositol 1-phosphate synthase genes could then be introduced intothe soybean genome (see U.S. Pat. No. 5,501,967) and result in newsoybean varieties displaying the instant phenotype.

[0074] Alternatively, gene silencing techniques such as antisenseinhibition technology (U.S. Pat. No. 5,107,065) and cosuppression (U.S.Pat. No. 5,231,020) may be employed to reduce the intracellularmyo-inositol 1-phosphate synthase activity in the cells of soybeanseeds. The instant specification teaches the sequence of the geneencoding the wild type soybean myo-inositol 1-phosphate synthase enzyme.The skilled artisan will readily appreciate how to make and how to usechimeric genes comprising all or part of the wild type sequence orsubstantially similar sequences to reduce myo-inositol 1-phosphatesynthase activity in soybean seeds.

[0075] Accordingly, the instant invention pertains to the identity,characterization and manipulation of a soybean enzyme that results inthe alteration of raffinose saccharide, sucrose, phytic acid andinorganic phosphate content of soybean seeds, thus leading to valuableand useful soybean products. As taught herein, reduction of myo-inositol1-phosphate synthase enzymatic activity by any of several means willresult in soybean seeds displaying the instant phenotype.

EXAMPLES

[0076] The present invention is further defined in the followingExamples, in which all parts and percentages are by weight and degreesare Celsius, unless otherwise stated. It should be understood that theseExamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain, and without departingfrom the spirit and scope thereof, can make various changes andmodifications of the invention to adapt it to various usages andconditions. Further, the present invention is not to be limited in scopeby the biological materials deposited, since the deposited materials areintended to provide illustrations of materials from which manyembodiments may be derived. All such modifications are intended to fallwithin the scope of the appended claims.

[0077] Usual techniques of molecular biology such as bacterialtransformation, agarose gel electrophoresis of nucleic acids andpolyacrylamide electrophoresis of proteins are referred to by the commonterms describing them. Details of the practice of these techniques, wellknown to those skilled in the art, are described in detail in [Sambrook,et al. (Molecular Cloning, A Laboratory Manual, 2nd ed. (1989), ColdSpring Harbor Laboratory Press]. Various solutions used in theexperimental manipulations are referred to by their common names such as“SSC”, “SSPE”, “Denhardt's solution”, etc. The composition of thesesolutions may be found by reference to Appendix B of Sambrook, et al.[supra].

Example 1 Discovery of a Soybean Gene Conferring Improved CarbohydrateComposition

[0078] Assays for Raffinose Saccharide Content

[0079] The following assays were used to analyze soybean seeds forraffinose saccharide content. Prior to each of the analytical measuresfor determination of raffinose saccharide content, seeds were allowed toair dry for at least one week to a moisture content of approximately 8%and then stored at approximately 40 to 500 and 40% relative humidity.Inventors' own measurements of many such air-dried samples indicatedthat the moisture content did not vary significantly from 8% (range of 7to 10%) when stored at these conditions.

[0080] For each individual raffinose saccharide assay, typically five toten soybeans from a given plant were ground in a Cyclotech 1093 SampleMill (Tecator, Box 70 S-26301, Hoganas, Sweden) equipped with a 100 meshscreen to yield a seed powder that was then analyzed. In most cases,raffinose saccharide content was determined for seed or derived productscontaining an “as is” moisture content of approximately 8%. In thesecases, “as is” values were converted to a dry basis (db) by dividingmeasurements by 0.92. For comparison among certain lines or amongcertain soy protein products, the ground seed powder was placed in aforced air oven at 45° until the samples reached constant weight (0%moisture) prior to analysis. Hence, all raffinose saccharidemeasurements reported within this specification are on a common drybasis unless otherwise specificied.

[0081] As describe below, three assays (“enzymatic”, “TLC”, and “HPLC”)were used to determine raffinose saccharide content of soybean seeds.All three were performed on seed powder derived from the aforementionedgrinding process.

[0082] In preparation for the “enzymatic” assay, after grinding eachseed sample, approximately 30 mg of the resultant powder was weighedinto a 13×100 mm screw cap tube and 1.6 mL of chloroform and 1.4 mL ofmethanol:water (4:3, v/v) was added. The precise powder weight of eachsample was recorded and used to adjust the following assay results forsample to sample weight differences. The tubes were then capped, placedin racks and shaken on a rotary shaker for 60 min at 1800 rpm at roomtemperature. After extraction, the contents of the tubes were allowed tosettle for 15 min. After settling, a 15 μL aliquot of the methanol:waterphase was placed in a well of a 96 well microtiter plate and dried at45° for 20 min. The dried wells were used as reaction vessels for thecoupled “enzymatic” assay which employed α-galactosidase and galactosedehydrogenase as described previously [Schiweck and Busching, (1969)Zucker 22:377-384; Schiweck and Busching, (1975) Zucker 28:242-243;Raffinose Detection Kit, Boehringer Mannheim GMBH, Catalog Number 428167] with modifications of the assay conditions. The modifications ofthe assay included addition of Bovine Serum Albumin (15 mg/mL) to theassay and a-galactosidase buffers, increasing the temperature and timeof the α-galactosidase incubation from room temperature to 45° and 30min, and increasing the time of the galactose dehydrogenase incubationfrom 20 min to 60 min, and using stachyose instead of raffinose for theα-galactoside standard. After incubation, the absorbance at 340 nm ofthe samples was determined on a BIO-TEK® Model EL340 Microplate reader.The amount of α-galactosides present in the samples was determined bycomparison to known quantities of the stachyose standard. To facilitatethe analysis of thousands of samples, enzymatic assays were replicatedonce. Lines that appeared to be low in raffinose saccharide content fromthe primary assay were subsequently reassayed in triplicate, beginningfrom the ground seed, if sufficient material was available. Lines whosecomposition was confirmed in the secondary assay were grown to maturityunder field conditions and seed from the field-grown plants were assayedagain. In cases where more specific information about the raffinosesaccharide and galactinol profile was required, low raffinose saccharidelines identified by the enzymatic assay were reassayed using the HPLCassay (described below).

[0083] To facilitate the rapid selection of low raffinose saccharidegermplasm, a thin layer chromatography “TLC” assay was developed. Forthis assay, about 60 mg of ground seed powder was placed into a 13×100mm screw top test tube, to which 1 mL of 4:3 (v/v) methanol:water and 1mL of chloroform were added. The tubes were capped, placed in racks andmixed on a rotary shaker for 60 min at 25 rpm at room temperature. Afterextraction the tubes were centrifuged at 2100 rpm for 5 min. A 4 μLsample was taken from the methanol:water layer and placed on a 20×20 cm‘Baker’ Silica Gel preadsorbent/channeled TLC plate with a 250 mmanalytical layer. Samples were allowed to dry at room temperature andthe plate was then placed in a TLC tank with a 3:4:4 (v/v/v) solution ofEthyl Acetate:Isopropanol:20% Acetic Acid (in water). The solution wasallowed to soak up the analytical channels for 10 cm, at which time theplate was removed and allowed to air dry in a fume hood. The plate wasthen sprayed with an aniline-diphenylamine reagent to identify thecarbohydrates in the sample. This reagent was prepared by mixing 1 mL ofaniline with 100 mL of acetone, 1 gram of diphenylamine, and 10 mL ofphosphoric acid. The plate was then placed in a 100° oven for 15 min todry and removed and allowed to cool down before reading the analyticalchannels. Stachyose and raffinose content in the soybean samples wereestimated by comparison to the elution pattern seen in pure standards.

[0084] A high performance anion exchange chromatography/pulsedamperometric assay, referred to herein as the “HPLC” assay, was used fordetermining the content of individual raffinose saccharides (e.g.,stachyose and raffinose), galactinol, and for confirming the results ofeither the enzymatic or TLC assays. Conditions for the grinding andextraction of the seed were identical to those used for the previous“enzymatic” assay. A 750 μL aliquot of the aqueous phase was removed anddried under reduced pressure at 80°. The dried material was thendissolved in 2 mL of water and mixed vigorously for 30 sec. A 100 μLaliquot was removed and diluted to 1 mL with water. The sample was mixedthoroughly again and then centrifuged for 3 min at 10,000×g. Followingcentrifugation, a 20 μL sample was analyzed on a Dionex™ PA1 columnusing 150 mM NaOH at 1.3 mL/min at room temperature. The Dionex™ PADdetector was used with E1=0.05 v, E2=0.60 v and E3=−0.60 v and an outputrange of 3 mA. Galactinol, glucose, fructose, sucrose, raffinose,stachyose and verbascose were well separated by the chromatographicconditions. The carbohydrate content of the samples was determined bycomparison to authentic standards.

[0085] Results obtained from the carbohydrate analyses were subjected toanalysis of variance using the software SuperANOVA (Abacus Concepts,Inc., 1984 Bonita Avenue, Berkeley, Calif. 94704). When appropriate,Fisher's Protected LSD was used as the post-hoc test for comparison ofmeans. In other comparisons, means were considered statisticallysignificant if the ranges defined by their standard errors (SEM's) didnot overlap. In cases where raffinose saccharide means were beingcompared to the mean of a control line, a mean was consideredsignificantly lower than that of the control if said mean was at leastthree standard deviations below that of the control mean.

[0086] Mutagenesis and Selection of Mutants

[0087] Approximately 130,000 seeds (22.7 kg) of LR13 (a line essentiallyidentical to Williams 82) were soaked in 150 L of tap water undercontinuous aeration for eight hours. Aeration was accomplished bypumping air through standard aquarium “airstones” placed in the bottomof the soaking vessel. Imbibed seeds were drained and transferred to 98L of a 2.5 mM N-nitroso-N-methylurea (NMU) solution buffered at pH 5.5with 0.1 M phosphate buffer under continuous aeration. Seeds remained inthe NMU solution for three hours and were then put through a series ofrinses to leach out the remaining NMU. For the first rinse, treatedseeds were transferred to 45 L of tap water for 1 min. For the secondrinse, seeds were transferred to 45 L of fresh tap water undercontinuous aeration for one hour. For the third rinse, seeds weretransferred to 45 L of fresh tap water under continuous aeration for twohours. For the fourth rinse, seeds were transferred to 45 L of fresh tapwater under continuous aeration. One half of the seeds were removed fromthe fourth rinse after two hours (sub-population 1) while the other halfof the seeds were removed from the fourth rinse after five hours(sub-population 2). After removal from the fourth rinse, seeds weredrained of exogenous water and spread out on cardboard sheets to dry offin the sun for one hour. The imbibed M1 seeds were then field planted(Isabela, Puerto Rico, USA) in rows spaced 46 cm apart at a density ofapproximately 14 seeds per foot within the rows and a depth of 2.5 cm.

[0088] Two pools of M2 seeds (from sub-populations 1 and 2) wereharvested in bulk from the M1 plants. Approximately 40,000 M2 seeds fromsub-population 1 and 52,000 M2 seeds from sub-population 2 were plantedat Isabela, Puerto Rico, USA. Within each sub-population, five pods fromeach of 3,000 M2 plants were harvested and bulked to obtain a bulk M3seed population. M3 bulks were planted at Isabela, Puerto Rico. Atmaturity, seed from 5000 M3 plants were harvested individually to obtain5000 M3:4 lines from each sub-population.

[0089] During the winter of 1991, a total of at least 8,000 M3:4 lineswere screened to measure the content of raffinose saccharides using theenzymatic method described above. In the initial screening, two lineswith decreased total raffinose saccharides were identified which provedheritable in a second, selfed generation. One of these lines, designatedLR33, had reduced levels of both stachyose and raffinose in comparisonto elite soybean cultivars grown as controls in the same environment. Incomparison to the average values for three elite cultivars grown in thesame environment, the stachyose content of LR33 was statisticallysignificantly lower. The soluble carbohydrate content of bulked seedsharvested from selfed lines derived from LR33 for three generations isshown in Table 1. TABLE 1 Soluble carbohydrates in mature seeds ofsoybean lines derived by selfing line LR33 GALACT- LINE STACHYOSERAFFINOSE INOL SUCROSE LR33 61 18 0 N.D.¹ 1ST-83 38 11 0 153 1991 Elite93 19 0 N.D.¹ (avg.) 2ST-88 51 13 0 209 1992 Elite 68 12 0 N.D.¹ (avg.)3ST-101 25  8 0 126 1993 Elite 76 17 0 N.D.¹ (avg.)

[0090] The subsequent lines (1ST-83, 2ST-88 and 3ST-101) were allderived by self pollination of LR33. Each of these lines had lowerstachyose contents than did control lines.

Example 2 Identification of the Physiological Defect Responsible for theLow Raffinose Saccharide Phenotype of LR33

[0091] Genetic Crosses Using LR33 as a Low Raffinose Saccharide Parent

[0092] In an effort to further reduce the total raffinose saccharidecontent of soybean seeds, genetic crosses were made between LR33 andLR28. Line LR28 is described in World Patent Publication WO93/07742.Briefly, it is also a low combined raffinose plus stachyose linediscovered in a screening proceedure very similar to that described inExample 1. The low raffinose and stachyose content is consistent fromone generation to the next. In addition, the galactinol content of themature seeds of line LR28 and lines derived from it by crossing to otherparents, is greatly elevated relative to wild type soybean seeds.

[0093] F1 plants from the cross of LR33 and LR28 were grown and selfpollinated to produce segregating F2 progeny. Seeds of LR28-derivedlines, elite cultivars and the segregating population resulting from thecross of LR28 and LR33 were planted in the same environment. F2:3 seedswere harvested from each F2 plant and screened for α-galactoside contentusing the enzymatic assay described above. Low α-galactoside selectionsfrom the preliminary screen were advanced to the HPLC assay to obtaincomplete raffinose saccharide profiles. Carbohydrate profiles fromtypical lines selected for low total α-galactoside content are shown inTable 2. TABLE 2 Soluble carbohydrates in mature seeds of soybean linesderived from crosses of line LR28 and LR33 and subsequent backcrosses toelite cultivars (LR33 and 5ST-1003 (an LR28 derived line) are includedas examples of the cross parents; 2242 as an elite cultivar control.)GALACT- LINE STACHYOSE RAFFINOSE INOL SUCROSE LR33 61 18 0 N.D.¹5ST-1441 57 18 0 N.D.¹ 5ST-1434  6 15 0 216 5ST-1309  0  5 0 287 2242 7518 0 168 5ST-1003 19  4 65  200

[0094] 5ST-1441, 5ST-1434, and 5ST-1309 were all derived from the LR33by LR28 cross. While line 5ST-1441 closely resembles the LR33 parent,surprisingly lines 5ST-1434 and 5ST-1309 are much lower in bothraffinose and stachyose than either parental line and do not contain theelevated galactinol level characteristic of line LR28.

[0095] In Vitro Assay of Activity of Enzymes in the Raffinose SaccharidePathway

[0096] In studies designed to find the cause of the unexpectedraffinose, stachyose and galactinol phenotype observed in some linesderived from the LR33 by LR28 cross, several enzyme activities andmetabolite pools were measured in soybean seeds harvested just beforethe physiologically mature stage during the period of rapid raffinosesaccharide biosynthesis. Seeds were removed from pods that had justbegun to yellow. Seeds from such pods that were also loosing green coloror just beginning to yellow themselves were chosen for assay of the lastthree enzymes in raffinose saccharide biosynthesis (See FIG. 1).

[0097] Seeds were removed from the pod, weighed to obtain fresh weight,then ground in a mortar and pestle in ten volumes of 50 mM HEPES-NaOH,pH 7 buffer which was also 5 mM in 2-mercaptoethanol. The ground sampleswere centrifuged at 10,000×g for 10 min and the supernatant was desaltedby passage through Sephadex G-25 which had been equilbrated in thegrinding buffer.

[0098] For the assay of galactinol synthase, 10 μL of the desaltedextract was added to 90 μL of the pH 7 HEPES buffer which was also 20 mMin myo-inositol, 10 mM in dithiothreitol, 1 mM in MnCl₂ and 1 mM inUDP-[¹⁴C]galactose (1.25 μCi μmol⁻¹). The assay mixture was incubatedfor 10 min at 25° then stopped by the addition of 400 μL of ethanol. Tothe stopped reactions was added 200 μL of Dowex AG-1×8 anion exchangeresin (BioRAD) and the mixture was shaken for 25 min. The resin wasremoved by centrifugation and the supernatant was taken forscintillation counting. Non-anionic radioactivity was taken as a measureof galactinol synthesis.

[0099] For the assay of raffinose synthase and stachyose synthase, 50 μLof the desalted extract was added to 50 μL of 25 mM HEPES buffer at pH 7that was also 10 mM in dithiothreitol, 10 mM in galactinol, and 40 mM insucrose for the assay of raffinose synthase or 40 mM in raffinose forthe assay of stachyose synthase. After a 1 h incubation at 25°, thereactions were stopped by the addition of 40 μL of ethanol and thenplaced in a boiling water bath for 1 min to precipitate proteins. Thereaction mixes were centrifuged to clear, the supernatant was passedthrough a 0.22 micron filter and then reduced to dryness under vacuum.The residue was re-dissolved in 0.5 mL of water and 20 μL were separatedon the Dionex HPLC system as described above for the quantitation ofraffinose and stachyose. Results of an experiment done using seedsharvested from field grown plants in August of 1994 are given in Table3. TABLE 3 Activities of galactinol synthase, raffinose synthase, andstachyose synthase in yellowing seeds of three soybean lines (Thewildtype control is line 1923. Enzyme activities are expressed as μmoleof product produced per gram of fresh seed weight per hour under theassay conditions. Values are mean ± standard deviation for fivereplicates.) GALACTINOL RAFFINOSE STACHYOSE LINE SYNTHASE SYNTHASESYNTHASE 1923  7.5 ± 4.1 0.10 ± 0.05 0.65 ± 0.18 LR28 16.5 ± 5.2 0.004 ±0.007 0.81 ± 0.24 LR28xLR33 22.6 ± 8.8 0.006 ± 0.008 0.87 ± 0.21

[0100] Of the three enzymes committed to raffinose and stachyosesynthesis, only raffinose synthase shows a clear decrease in activity inthe mutant, low raffinose saccharide lines in comparison to wild type. Amutation causing decreased activity of raffinose synthase is consistentwith the position of that enzyme in the biosynthetic pathway and theaccumulation of galactinol observed in lines which carry only LR28 asthe source of the low raffinose saccharide phenotype. Galactinol andsucrose are the two substrates for raffinose synthase and bothmetabolites accumulate in LR28 containing lines (see FIG. 1 and Table2).

[0101] Despite the lower levels of stachyose, raffinose and galactinolobtained when LR33 lines were crossed with LR28 lines, the reducedraffinose synthase activity conferred by the LR28 mutation is the onlyaltered activity. While decreased galactinol synthase activity seemed alikely candidate for the lesion in the LR33 line due the decreasedgalactinol content conferred by the mutation when in combination withLR28, there is no decrease in the measured, in vitro activity of thatenzyme.

[0102] Assay of the Free myo-inositol Content of Maturing Soybean Seeds

[0103] While there was no decrease in galactinol synthase in vitro inthe LR33 by LR28 cross, the possibility that the in vivo activity ofgalactinol synthase in maturing seeds of LR33 derived lines is limitedby the availability of one of its substrates was checked. The supply ofUDP-galactose to galactinol synthase could be decreased by mutations inUDP-glucose 4′ epimerase and the supply of myo-inositol could be limitedby mutation effecting either its synthase or the specific phosphatasethat produces the free inositol form (FIG. 1). The myo-inositol pool waschecked by assaying for the total free myo-inositol content in wild typeand LR33 derived seeds. Three soybean lines, a wild type cultivar A2872,and two LR33×LR28-derived lines, 5ST-1434 and 5ST-1309, were grown in agrowth room under 16 h days with a day/night temperature regime of30°/22°. Seeds were taken from pods that had just begun to yellow andthat were themselves turning from very light green to yellow. Threeseeds from each line were ground in 8 mL of 80% methanol. The mixturewas centrifuged at 12,000×g for 15 min and the supernatant decanted to a50 mL flask. The extraction was repeated twice and the supernatantscombined. The combined supernatants were reduced to dryness under vacuumat 40° and the residues re-dissolved in 1 mL of water. The re-dissolvedextracts were deionized by passage through 3 mL of mixed bed ionexchange resin (BioRad AG501×8) and the through flow plus 4 mL of washwas again reduced to dryness. The residue was re-dissolved in 500 μL ofwater and a 70 μL aliquot was separated by HPLC on a Zorbax Amino column(DuPont) eluted at 1 mL min⁻¹ with 70% acetonitrile. Sugars in thecolumn eluate were detected by differential refractive index. Separationof standard mixtures of sucrose and myo-inositol showed that inositolemerges later and only partially separated from sucrose. For analysis ofthe seed extracts, 1 mL of eluate following the sucrose peak was trappedfor re-injection on the Dionex PAD system described in Example 1. In theDionex system, myo-inositol emerges well before sucrose and could becleanly separated from sucrose contaminating the Zorbax Amino columnfraction. By comparison to external standards of myo-inositol, the 5 μLinjection from the wild type 2872 line contained 3.2 nmoles ofmyo-inositol while 5 μL injections from 5ST-1434 and 5ST-1309 contained0.39 and 0.23 nmoles respectively.

[0104] A second experiment was performed to further characterize thedifference in free myo-inositol content of wild type and LR33 derivedseeds. Seven single seeds from a second control line (2242) and from theLR33 derived line 5ST-1434 were harvested according to the maturitycriteria described above. The seeds were individually weighed, placed in1.5 mL microfuge tubes, crushed with a spatula, frozen on dry ice andlyophilized. The dry residue was weighed and 1 mL of 80% methanol whichcontained 1 μmole of trehalose was added to act as a myo-inositolretention marker on the Zorbax amino column and as an internal standard.The extraction media was heated for 1 h at 60°, re-ground with a smallpestle in the microfuge tube and centrifuged to pellet the insolublematerial. The supernatant was transferred to a second microfuge tubecontaining about 100 μL of mixed bed resin, the mixture was brieflyshaken and 20 μL of the solution above the resin was removed and takento dryness under vacuum. The residue was re-dissolved in 110 μL totalvolume and 55 μL were injected onto the Zorbax Amino column run asdescribed above. The trehalose peak was trapped and 20 μL of the columnfraction was re-injected on the Dionex system. The myo-inositol peak wasquantitated by comparison to the trehalose internal standard. Themean±standard deviation for the wild type myo-inositol content was2.18±0.98 μmole (g dry wt)⁻¹ while the 5ST-1434 seeds contained0.77±0.32 μmole (g dry wt)⁻¹. Both experiments indicate that seeds whichcarry the LR33 mutation contain less myo-inositol during the period ofrapid raffinose saccharide synthesis. In neither experiment was thetotal myo-inositol pool totally absent. Such experiments can onlymeasure the total seed pool of metabolites however and myo-inositol hasother fates which could further limit its use by galactinol synthase(FIG. 1).

[0105] The Effect of myo-inositol Perfusion on the in Vivo Labeling ofRaffinose Saccharides

[0106] To ascertain whether or not the observed decrease in freemyo-inositol content of the LR33 derived seeds might be the cause of thedecreased raffinose saccharide content at maturity, tissue slice feedingstudies were performed. Four seeds each from wild type line 2242 andLR33-derived line 5ST-1434 were harvested by the maturity criteriondescribed above. The seed coats were removed and the cotyledons rinsedin 5 mM potassium phosphate buffer at pH 5.5 then sliced intoapproximately I mm slices and again rinsed with buffer. The tissueslices were divided into two groups. One group was immersed in potassiumphosphate buffer and the second group was immersed in the potassiumphosphate buffer containing 50 mM myo-inositol. The tissue slices werevacuum-infiltrated and incubated for 30 min at room temperature. Afterthe pre-incubation period, 5 μCi of ¹⁴C-glucose was added to eachgrouping and the tissue was again vacuum-infiltrated. Ten tissue slicesfrom each group were taken at 2 h and at 8 h after addition of thelabeled glucose. The tissue slices were placed in tarred 1.5 mLmicrofuge tube to obtain fresh weight and ground in 300 μL of 80%methanol. The tubes were centrifuged, the supernatant removed to asecond tube, the extraction was repeated twice more and the supernatantswere combined. The combined supernatants were reduced to dryness undervacuum, re-dissolved in 50% acetonitrile and separated on the Zorbaxamino HPLC system. Fractions were collected for scintillation countingat 15 second intervals through the region of the chromatogram containingglucose, sucrose, raffinose and galactinol and at 1 min intervalsthrough the region containing stachyose. Radioactive fractionscorresponding to the sugar standard peaks were grouped for analysis. Theresults are expressed as per cent of the radiolabel in the four productsugars in Table 4. TABLE 4 Radiolabel in sucrose, galactinol, raffinoseand stachyose expressed as per cent of label in those sugars combinedfor control and an LR33 derived line with and without pre-incubationwith myo-inositol WILD TYPE TISSUE SLICES 5ST-1434 TISSUE SLICES 2 h 2h + inos 8 h 2 h + inos 2 h 2 h + inos 8 h 8 h + inos SUCROSE 52.4 33.441.0 23.8 86.8 43.5 79.8 21.8 GALACTINOL 13.4 24.8 8.0 11.1 3.2 27.6 1.59.5 RAFFINOSE 19.8 20.7 17.3 17.7 4.5 15.2 3.1 13.0 STACHYOSE 14.4 21.033.6 47.4 5.4 13.7 15.9 55.6

[0107] Both wild type and 5ST-1434 tissue slices convert label fromsupplied ¹⁴C-glucose to sucrose and then into galactinol, raffinose, andstachyose. Without the addition of exogenous myo-inositol, the linecontaining the LR33 mutation (5ST-1434) converts very little label intothe raffinose saccharides (20.5% after 8 h) in comparison to the wildtype line (58.9% after 8 h). Both genotypes respond to exogenousmyo-inositol by converting more of the supplied label to raffinosesaccharide sugars. With the addition of myo-inositol the two genotypesbecome essentially equal in their ability to convert the supplied labelfirst into galactinol and then into raffinose and stachyose. The rate ofconversion is increased even in the wild type line in comparison to thenon-infused control.

[0108] It appears that the supply of myo-inositol to galactinol synthaseis always a limitation to the rate at which raffinose and stachyose canbe synthesized. The presence of the LR33 mutation makes that limitationmuch greater.

[0109] The labeling pattern is surprising in that there is no buildup oflabel in galactinol as would be expected if the LR28 mutation inraffinose synthase were still effective in slowing flux through thatstep in the combined mutant lines. While the addition of myo-inositolcan be expected to increase flux through galactinol synthase togalactinol, the label should have accumulated there due to the decreasedactivity in raffinose synthase. The absence of that accumulation callsinto question either the effectiveness or the presence of the LR28mutation in this line.

[0110] The Influence of the LR33 Mutation on Seed Phytic Acid andInorganic Phosphate Levels

[0111] The above results suggest that the LR33 mutation resides beforefree myo-inositol in the pathway shown in FIG. 1. To further limit thepossible site of the mutation, other metabolites in that portion of thepathway were measured.

[0112] Since soybean seeds contain phytic acid at levels of 20 to 30μmoles g⁻¹ at maturity and since 50 to 70% of the total seed phosphateis contained in phytic acid [Raboy et al. (1984) Crop Science24:431-434], it seemed likely that mutations effecting myo-inositolsynthesis might also effect seed phytic acid and inorganic phosphatelevels. The levels of phytic acid and inorganic phosphate in three wildtype soybean lines and three lines that contain the LR33 mutation wereassayed in dry seeds by a modification of the method described by Raboy[supra]. Approximately twenty seeds from each line were ground in asmall impact mill and 100 mg of the resulting powder was weighed into a15 mL screw cap tube. Five mL of 0.4 N HCl with 0.7 M Na₂SO₄ was addedto the powder and the mixture was shaken overnight on a rocker platform.The tubes were centrifuged at 3900×g for 15 min and 2 mL of thesupernatant was removed to second, glass tube. Two mL of water and 1 mLof 15 mM FeCl₂ were added and the tubes were heated for 20 min in aboiling water bath. The tubes were centrifuged as above to precipitatethe iron:phytic acid complex and the supernatant was discarded. Thepellets were resuspended in 2 mL of 0.2 N HCl and heated for 10 min inthe boiling water bath. The precipitate was again removed bycentrifugation and re-suspended in 2 mL of 5 mM EDTA.

[0113] Fifteen μL of the EDTA suspension was taken for wet ashing toobtain phytic acid phosphate. To each tube containing the 15 μL aliquotwas added 10 μL of a 10% solution of calcium nitrate. The water wasallowed to evaporate and the residue was heated in a flame until a whiteash remained in the tube. The ash was dissolved in 0.3 mL of 0.5 N HCLand heated at 90° for 20 to 30 min. Acid molybdate reagent (0.7 mL of0.36% ammonium molybdate and 1.42% ascorbic acid in 0.86 N sulfuricacid) was added and the color was allowed to develop for 1 h beforereading at 820 nm.

[0114] Inorganic phosphate was determined by adding 0.3 mL of 0.5 N HCland 0.7 mL of the phosphate color reagent to either 20 or 40 μL aliquotsof the initial 5 mL extract. Potassium phosphate standards weredeveloped at the same time. The experiment was repeated three times andthe results are given in Table 5. TABLE 5 Seed phosphate in inorganicphosphate and in phytic acid expressed as μmoles phosphate g⁻¹ (Valuesare mean ± standard deviation where more that two replicates wereobtained or the average value when two replicates were obtained.) SEEDREPLI- INORGANIC PHYTIC ACID LINE GENOTYPE CATES PHOSPHATE PHOSPHATE2872 WILD TYPE 7 2.7 ± 3   125 ± 8.4  1923 WILD TYPE 1 2.0 126 1929 WILDTYPE 2 1.5 155 5ST-1309 LR33 4 76.0 ± 5.7 62.5 ± 12.4 GxE-117 LR33 736.7 ± 6.4 55.8 ± 13.9 GxE-76 LR33 6 32.2 ± 3.5 72.8 ± 14  

[0115] The LR33 mutation also causes a decrease in seed phytic acidlevel (expressed as phosphate in the phytic acid fraction) of about twofold with a concomitant increase in the seed inorganic phosphate contentof about 15 to 25 fold depending upon the genetic background in whichthe mutation is contained. Based on the pathway in FIG. 1, the decreasedphytic acid content, along with the decreased free myo-inositol contentare most likely to be caused by a mutation decreasing the activity ofmyo-inositol 1-phosphate synthase.

[0116] The exact cause of the increase in the inorganic phosphatecontent is not known, however it has long been assumed that phytic acidfunctions as a phosphate storage form in seeds and other parts of theplant. It is possible that when the preferred storage platform(myo-inositol) is unavailable, incoming inorganic phosphate has no othermajor fate and simply accumulates.

Example 3 Identification of the Seed Phenotype of the Homozygous LR33Line

[0117] Several additional soybean lines were analyzed for seed inorganicphosphate. One hundred mg of ground seed was extracted with 1 mL of hotwater and centrifuged to remove the insoluble matter. The supernatantwas extracted with 100 μL of methylene chloride to remove lipid materialand the remainder of the aqueous extract was made to 10% withtrichloroacetic acid. The solution was centrifuged to remove proteinsand 5 μL of the supernatant was analyzed for inorganic phosphate asdescribed in Example 2. Table 6 gives the results of those assays. TABLE6 The inorganic phosphate content (μmole g⁻¹) of seeds from wild type,LR28, LR33 and LR28xLR33 soybean plants LINE GENOTYPE INORGANICPHOSPHATE A2872 wildtype 12.7 LR28 LR28 12.7 LOW2 LR28 12.9 5ST-1190LR28 14.4 5ST-1191 LR28 14.0 5ST-1441 LR33 23.4 5ST-1309 LR28xLR33 117.65ST-1310 LR28xLR33 113.9 5ST-1433 LR28xLR33 74.2 5ST-1434 LR28xLR33 57.1GxE-117 LR28xLR33 74.3 GxE-305 LR28xLR33 70.7 GxE-76 LR28xLR33 79.1GxE-77 LR28xLR33 58.4

[0118] All of the lines which contain the LR28 mutation alone haveinorganic phosphate levels equal to the wild type controls. Line5ST-1441 was derived from LR33 (Example 2 above) and has the moderatereduction in stachyose that was originally associated with the mutation.5ST-1441 has only a slightly increased inorganic phosphate content.Despite the fact that neither parent is high in inorganic phosphate, allthe lines derived form the cross of LR33 by LR28 have greatly elevatedlevels of inorganic phosphate.

[0119] The intermediate inorganic phosphate phenotype of line 5ST-1441that contains only the LR33 mutation was unexpected. It may indicatethat both the LR28 and the LR33 mutation must be present to produce thelow phytic acid/high inorganic phosphate phenotype. If however the twomutations are in fact in the structural genes encoding the twoactivities that seem to be decreased, they are not related in themetabolic pathway in a manner that would suggest an additive effect onalteration of phytic acid synthesis. If the mutation in LR33 effectseither free or total myo-inositol production, it alone should beresponsible for the low phytic acid and resultant high inorganicphosphate phenotype.

[0120] An alternate explanation may be that the line 5ST-1441 is nothomozygous for the LR33 mutation and that analysis of the bulk seedgives only the average phenotype for the wild type, homozygous mutantand heterozygous seeds. To check this possibility, thirteen single seedsfrom the bulk sample of 5ST-1441 seed were weighed, ground and extractedwith hot water. Protein was not precipitated and inorganic phosphate wasmeasured as before. The results are shown in Table 7. TABLE 7Approximate inorganic phosphate content (μmoles g⁻¹) for thirteenindividual seeds form line 5ST-1441 (Actual values are elevated incomparison to the results of Table 6 due to the presence of protein inthe samples.) SEED NUMBER INORGANIC PHOSPHATE 5ST-1441-a 21.9 5ST-1441-b30.4 5ST-1441-c 25.4 5ST-1441-d 81.1 5ST-1441-e 29.7 5ST-1441-f 25.75ST-1441-g 25.5 5ST-1441-h 30.8 5ST-1441-i 28.2 5ST-1441-j 123.55ST-1441-k 27.9 5ST-1441-l 115.9 5ST-1441-m 27.6

[0121] Seeds lettered d, j, and 1 have about three told more inorganicphosphate than the remained ten seeds analyzed. The ratio approximatesthat expected from a segregating population of seeds in which the mutantphenotype of high inorganic phosphate is recessive and due to the actionof a single gene. To check this hypothesis, twenty seeds from the bulksample of 5ST-1441 were imbibed in water at room temperature for 6 h.Excess water was blotted away and a sample of the cotyledons at the endaway from the embryonic axis was cut off. The tissue pieces wereweighed, placed in 1.5 mL microfuge tubes and ground in sufficient 70%methanol to give 10 mg fresh wt per 100 μL of extracted volume.Phosphate in the supernatant present after centrifugation was measuredas described above and the results are shown Table 8. TABLE 8 Theinorganic phosphate content of 20 partial seeds from a segregatingpopulation of seeds from the line 5ST-1441 (Results are expressed asμmoles per g fresh weight (μmole gfwt⁻¹).) SEED NO. PO₄ (μmole gfwt⁻¹) 14.9 2 4.3 3 5.3 4 40.1 5 6.1 6 5.0 7 37.1 8 9.9 9 5.9 10 3.8 11 26.9 124.7 13 5.5 14 3.9 15 5.2 16 17.7 17 5.9 18 4.9 19 2.5 20 28.9

[0122] Seeds 4, 7, 11, 16 and 20 were planted in pots in a growth room.Seeds 7, 11, 16, and 20 survived and were grown to maturity under thegrowth conditions described in Example 2. Bulk seeds from the matureplants and two wild type controls (A2872) were harvested after dry downand twenty seeds from each plant were ground in bulk for analysis ofsoluble carbohydrates and phytic acid using the methods described inExamples 1 and 2. The total seed phytic acid content was calculated asthe (phytic acid phosphate content)/6. The results are shown in Table 9.TABLE 9 Soluble carbohydrate content and phytic acid content (expressedas μmole g⁻¹) for four plants from a segregating population of seedscarrying the LR33 mutation and selected for elevated inorganic phosphatein partial seed analysis along with two control plants (Phytic acidvalues are the average of two replicates.) LINE PHYTIC ACID STACHYOSERAFFINOSE GALACTINOL SUCROSE A2872 32.3 71 19 3 166 A2872 30.8 67 24 0144 LR33-7 23.7 40 16 0 155 LR33-11 8.8 4 13 0 212 LR33-16 9.5 4 13 2209 LR33-20 7.6 4 11 0 208

[0123] While the soluble carbohydrate phenotype of the plant grown fromseed number 7 (LR33-7) is very similar that ascribed to the LR33mutation as shown in Table 1, the other plants have much lower levels ofthe raffinose saccharides and have much higher levels of sucrose. Theselevels of soluble carbohydrate are very similar to the phenotypeascribed to some plants in the mutant combination LR28×LR33 in Table 2.The most probable explanation for this discrepancy is that the bulk seedanalyzed to give the data in Table 1 came from plants segregating forthe LR33 mutation rather than plants homozygous for the mutation. Whenplants homozygous for the mutation are selected, as was done in the caseof plants 11, 16 and 20 in this example, the true, homozygous seedphenotype is observed. The previous assumption that both the LR28 andthe LR33 mutations needed to be present to produce the combination oflow raffinose, stachyose and galactinol was incorrect. While lineshomozygous for the LR33 mutation were not obtained from selfing theoriginally identified mutant line, they were obtained from segregantsarising from the cross of that line with LR28. While some of the progenyof the cross likely do contain both mutations, the phenotype from theLR33 mutation is dominant to that arising from the LR28 mutation. Thatdominance stems primarily from the location of the LR33 mutationupstream in the carbon flow from the LR28 mutation (see FIG. 1 andExample 2).

[0124] The reason that the homozygous LR33 phenotype remained elusivemay be due to germination problems encountered with the original mutantline. In bulk field segregation conditions the loss of 25% (thatfraction that would have been homozygous for the mutation) of theplanted seed from selfing LR33 could have been missed. Harvested plantsfrom the bulk population would have then been depleted in homozygotesbut the mutant gene would be retained in the population in theheterozygous state. We surmise that out crossing to a genetic backgroundmore conducive to allowing LR33 homozygotes to emerge in fieldconditions allowed recovery of the homozygote. That the initial outcrosses were to lines carrying another mutation in the raffinosesaccharide biosynthetic pathway were incidental to the original intentof the cross and not essential either for the superior phenotype or theimproved field emergence.

[0125] The LR33 mutation is thus capable of producing soybean plantsthat produce seeds with less than 5 μmoles of stachyose per gram ofseed, less than 20 μmoles of raffinose per gram of seed, more than 200μmoles of sucrose per gram of seed and less than 10 μmoles of phyticacid phosphate per gram of seed.

Example 4 Soluble Carbohydrate and Phytic Acid Content of a Soybean LineContaining the LR33 Mutation

[0126] The low raffinose saccharide lines LR33 and LR28 were crossed andsegregating F2 plants were selected for low levels of raffinose,stachyose and galactinol in the seed of the F2 plants by the HPLC assaydescribed in Example 1. Selected lines derived from this cross were thencrossed to elite cultivar parents and the progeny of those crosses wereselected in the F2 generation by the same process. Among these linescontaining low levels of raffinose saccharides, several were chosen foradvancement based on the agronomic characteristics of the vegetativeplant. One such line, designated 4E76, was subsequently test crossed toanother elite soybean cultivar and single seeds from the selfed F1 plantderived from that cross were analyzed for soluble carbohydrates and forinorganic phosphate content. The seeds fell into two classes, those withvery low inorganic phosphate and wild type levels of raffinose plusstachyose, and a smaller class of seeds with elevated levels ofinorganic phosphate and very low levels of raffinose plus stachyose. Theratio of these two classes was very near the 3:1 ratio expected of therecessive LR33 mutation. The was no evidence of segregation of LR28which is the other mutation effecting raffinose saccharides that waspresent in the original cross. Line 4E76 thus represents the LR33mutation in a selected elite cultivar background. The line was grown ina total of nine environments over two years along with selected elitecheck lines for carbohydrate analysis. A more limited set of samplesfrom three environments was analyzed for phytic acid content. Thecarbohydrate data is shown in Table 10 and the phytic acid data in Table11. TABLE 10 Mean, standard deviation (SD) and two standard deviations(2SD) of the mean for the stachyose, raffinose and sucrose content ofLR33-containing line 4E76 and thirteen elite cultivar checks (All valuesare expressed as μmoles g⁻¹ seed weight.) STACHYOSE RAFFINOSE SUCROSELINE MEAN SD 2SD MEAN SD 2SD MEAN SD 2SD 4E76 2.4 0.5 1.0 6.9 0.7 1.4249 42.9 85.8 A2514 44.8 2.7 5.4 13.0 2.6 5.2 138 9.6 19.2 A2396 47.81.1 2.2 9.6 0.9 1.8 158 9.6 19.2 A1923 50.2 2.8 5.6 8.6 0.5 1.0 148 9.418.8 A2506 51.0 3.7 7.4 16.4 4.0 8.0 161 28.2 56.4 A3322 54.2 7.3 14.614.0 2.7 5.4 147 27.1 54.2 A2923 56.4 2.2 4.4 13.6 1.7 3.4 125 9.2 18.4A2234 57.2 9.1 18.2 16.6 3.4 6.8 145 21.2 42.4 A1923 57.5 4.5 9.0 18.53.4 6.8 156 29.8 59.6 A3313 61.0 9.7 19.4 14.4 3.9 7.8 172 37.9 75.8A1662 61.6 6.2 12.4 13.8 3.4 6.8 127 21.4 42.8 A3935 62.8 9.2 18.4 11.62.1 4.2 184 38.7 77.4 A2833 63.8 8.6 17.2 15.4 2.4 4.8 150 38.7 77.4A3510 68.8 9.4 18.8 14.2 1.9 3.8 160 33.0 66.0

[0127] TABLE 11 Mean, standard deviation and two standard deviations ofthe mean for the phytic acid content of the LR33-containing line 4E76and the elite cultivar check A2872. (All values are expressed as μmolesg⁻¹ seed weight and represent data from three environments.) PHYTIC ACIDLINE MEAN SD 2SD 4E76 12.3 2.4 4.8 A2872 20.9 0.9 1.8

[0128] In comparison to elite soybean cultivars that are typical ofcommercial soybeans, the LR33-derived line is eight to nine fold lowerin the mass of total raffinose saccharide per gram of seed weight. Themass of phytic acid is also decreased by about 40%.

[0129] In comparison to elite soybean cultivars, the LR33-derived linesare lower in the mass of both raffinose and stachyose. While raffinosecontent is only slightly lower than values seen in elite lines,stachyose content is very greatly reduced. It is known that thedetrimental effect of soybean raffinose saccharide content on energy useefficiency when soybean meal is fed to monogastric animals is due to thepoor digestibility of the α-galactosidic bond. Accordingly, a decreasein combined raffinose plus stachyose content is an appropriate measureof increased digestibility. In fact, this measurement may evenunderestimate the effect of the instant phenotype since stachyosecontains two moles of the α-galactosidic bond per mole of sugar.

[0130] The absolute raffinose saccharide content of mature soybeans isknown to vary in response to environmental factors. The effect of themutation present in LR33-derived lines is of sufficient magnitude torender the combined raffinose plus stachyose content of such lines below14.5 μmol/g seed weight and should always remain far below that of wildtype soybeans grown in the same environment.

[0131] The phytic acid content of mature soybean seeds is also known tovary in response to environmental factors, primarily due to variation oflevels of available phosphate in soil. Once again, the mutation presentin LR33-derived lines maintains total seed phytic acid content below 17μmol/g across all growth environments.

Example 5 Molecular Identification of the LR33 Mutation

[0132] The evidence from analysis of metabolites in the LR33 derivedlines presented in Example 2 strongly suggests that the LR33 mutationcauses a decrease in the ability of the seed to produce myo-inositol.

[0133] In plants as well as other organisms, myo-inositol is producedsolely by a pathway which begins with the conversion ofglucose-6-phosphate to myo-inositol 1-phosphate in a reaction catalyzedby myo-inositol 1-phosphate synthase and ends with the hydrolysis of the1-phosphate by a specific myo-inositol-phosphate phosphatase (see FIG.1; Loews, F.A. In: Inositol Metabolism in Plants (1990) Wiley-Liss, NewYork, pp 13-19]). Since the phytic acid has as its probable precursormyo-inositol 1-phosphate and since the level of phytic acid is decreasedby the LR33 mutation, the gene and gene product formyo-inositol-phosphate synthase was characterized in both wild type andLR33 mutant plants.

[0134] cDNA Cloning of the Wild Type Soybean myo-inositol 1-phosphateSynthase

[0135] A cDNA library was made as follows. Soybean embryos (ca. 50 mgfresh weight each) were removed from their pods and frozen in liquidnitrogen. The frozen embryos were ground to a fine powder in thepresence of liquid nitrogen and then extracted by Polytronhomogenization and fractionated to enrich for total RNA by the method ofChirgwin et al. ((1979) Biochemistry 18:5294-5299).

[0136] The nucleic acid fraction was enriched for poly A⁺ RNA by passingtotal RNA through an oligo-dT cellulose column and eluting the poly A⁺RNA with salt as described by Goodman et al. ((1979) Meth. Enzymol.68:75-90). cDNA was synthesized from the purified poly A⁺ RNA using cDNASynthesis System (Bethesda Research Laboratory, Gaithersburg, Md.) andthe manufacturer's instructions. The resultant double-stranded DNA wasmethylated by Eco RI DNA methylase (Promega) prior to filling in itsends with T4 DNA polymerase (Bethesda Research Laboratory) and blunt-endligation to phosphorylated Eco RI linkers using T4 DNA ligase(Pharmacia). The double-stranded DNA was digested with Eco RI enzyme,separated from excess linkers by passage through a gel filtration column(Sepharose CL-4B), and ligated to lambda ZAP vector (Stratagene)according to manufacturer's instructions. Ligated DNA was packaged intophage using the Gigapack™ packaging extract (Stratagene) according tomanufacturer's instructions. The resultant cDNA library was amplified asper Stratagene's instructions and stored at −80°.

[0137] Following the instructions in the Lambda ZAP Cloning Kit Manual(Stratagene), the cDNA phage library was used to infect E. coli XL1cells and a total of approximately 300,000 plaque forming units wereplated onto six 150 mm diameter petri plates.

[0138] Duplicate lifts of the plates were made onto nitrocellulosefilters (Schleicher & Schuell, Keene, N.H.). The filters wereprehybridized in 25 mL of hybridization buffer consisting of 6×SSPE,5×Denhardt's solution, 0.5% SDS, 5% dextran sulfate and 0.1 mg/mLdenatured salmon sperm DNA (Sigma Chemical Co.) at 60° for 2 h.

[0139] The blocked filters were then hybridized to a radiolabelled probemade from a cDNA from Arabidopsis thaliana which had been identified asa myo-inositol-1-phosphate synthase by homology to yeastmyo-inositol-1-phosphate synthase [Johnson, M.A. (1994) Plant Physiol.105:1023-1024]. The Arabidopsis clone was obtained from the ArabidopsisBiological Resource Center, DNA Stock Center, 1060 Carmack Road,Columbus, Ohio 43210-1002, clone number 181C18T7113E9T7. The 1.2 kB cDNAinsert was removed from the vector DNA by digestion with Sal I and Not Ifollowed by agarose gel purification of the DNA fragment. The purifiedfragment was labeled with [³²P]dCTP with a random primer labeling kit(Bethesda Research Laboratory). The filters were allowed to hybridizeovernight under the same conditions as described for pre-hybridization.Excess radiolabel was washed from the filters in 0.6×SSC containing 0.1%SDS. Two washes of about 10 min each in 0.2×SSC, 0.1% SDS at 60° werethen applied to remove non-specifically bound label and the filters wereused to expose photographic film in an overnight exposure. Approximately200 positive signals were observed. Six were purified by excision of thearea around the signal, re-plating the phage and re-screening as above.Two clones were excised to phagmids and used to infect E. coli to obtainplasmid clones using the protocols described by the manufacturer(Strategene). Of the two clones, one designated p5bmi-1-ps was sequencedusing Applied Biological Instruments methodology and equipment. Thenucleotide sequence of the cDNA insert in p5bmi-1-ps is shown in SEQ IDNO: 1 and the deduced amino acid sequence encoded by that sequence inSEQ ID NO: 2.

[0140] cDNA Cloning of myo-inositol 1-phosphate Synthase from ImmatureSeeds of LR33 Soybeans

[0141] Seeds from the LR33 plants numbered 11, 16 and 20 and describedin Example 3 (see Table 8) were harvested at about 50% through the seedfilling period, removed from the pod and stored frozen at −80°. For mRNAisolation, 2 g of frozen seed from the bulked seed population wereground in liquid nitrogen in a mortar and pestle.

[0142] The frozen powder was again ground in 10 mL of total RNAextraction buffer which consisted of 10 mM Tris-HCl, pH 9, 10 mM EDTA,0.5% CTAB (cetyltrimethyl ammonium bromide), 0.8 M NaCl, 1%2-mercaptoethanol, and 2% polyvinylpyrrolidone. Water for all reagentswas treated with 0.05% diethylpyrocarbamate for 30 min then autoclaved.The tissue slurry was transferred to a 15 mL polypropylene tube andcentrifuged at 5,500×g for 15 min. The supernatant was passed throughMiracloth (Calbiochem) into a second polypropylene tube and 0.3 volumeof chloroform was added. The phases were separated by centrifugation at5,500×g for 10 min and the upper phase transferred to anotherpolypropylene tube to which was added 1.5 volumes 10 mM Tris-HCl pH 9,10 mM EDTA, 0.5% CTAB and 0.1% 2-mercaptoethanol. After a 30 minincubation at room temperature the nucleic acids were precipitated bycentrifugation at 5,500×g for 20 min.

[0143] The nucleic acids were re-dissolved in 0.4 mL of 1 M NaClcontaining 0.1% 2-mercaptoethanol. After extraction with one volume of1:1 phenol:chloroform, the nucleic acids were precipitated with twovolumes of ethanol.

[0144] mRNA was purified from this nucleic acid fraction using the mRNApurification kit from Pharmacia. Approximately 12 μg of mRNA wasobtained. Thirteen ng of the polyadenylated mRNA was used as templatefor amplification from oligo-dT using a GeneAmp® RNA-PCR kit (PerkinElmer Cetus, part no. N808-0017). The reverse transcriptase reaction wasrun for 30 min at 42° C. For the PCR amplification, Vent™ DNA polymerase(New England Biolabs) was substituted for the DNA polymerase supplied bythe kit manufacturer and an additional 2 μL of 100 mM magnesium sulfatewas added to each 100 μL reaction. The 5′ primer had the sequence shownin SEQ ID NO: 3 and consists of bases 57 to 77 in SEQ ID NO: 1 with theadditional bases 5′-GGGAATTCCATATG-3′ added to encode an Nde I site inthe primer with eight additional 5′ bases to enhance the restrictionenzyme activity against the sequence.

[0145] The 3′ primer had the sequence shown in SEQ ID NO: 4 and consistsof the reverse complement of bases 1566 to 1586 in SEQ ID NO: 1 with theadditional bases 5′-AAGGAAAAAAGCGGCCGC-3′ added to provide a Not I sitein the primer and ten additional bases to enhance restriction digestion.The PCR reaction was run for 35 cycles at a 52° annealing temperatureand 1.5 min extension time. A product of about 1550 base pairs wasobtained and purified by passage through an Amicon 50 microfuge filterfollowed by extraction with an equal volume of 1:1 phenol:chloroform,extraction of the upper layer of the phenol:chloroform separation withone volume of chloroform and precipitation with ethanol. Five μg of theresulting, clean PCR product was digested overnight at 37° with both NdeI and Not I. The restriction enzyme digest was de-proteinized by theabove described phenol:chloroform extraction procedure and ligated into2 μg of pET24aT7 expression vector (Novogen) that had also been digestedwith Nde I and Not I and treated with calf intestine alkalinephosphatase to hydrolyzed the terminal phosphates. The ligation mixturewas used to transform electocompetant DH 10B E. coli cells andtransformants were selected by growth on plates containing 30 mg 1⁻¹kanamycin. Eighteen single colonies from the transformation plate werepicked and placed in 100 μL of sterile water. Forty μL of the cell mixwas used as the DNA template in a PCR reaction run with Taq™ polymerase(Perkin Elmer) using the primers and PCR conditions that were initiallyused to isolate the cDNA insert. Six colonies served as template toamplify a product of the correct size. The remaining 60 μL of the cellmix from these clones was grown in overnight culture and plasmid DNApreparations were made from each clone. The purified plasmid from eachof the six clones was used to transform electrocompetant DE 3 E colicells.

[0146] Functional Expression of the myo-inositol-1-phosphate Synthasefrom Wildtype and LR33 Soybeans in E coli

[0147] The wild type soybean myo-inositol 1-phosphate synthase wasplaced into the pET24aT7 expression vector by PCR amplification ofp5bmi-1-ps using the primers in SEQ ID NO: 3 and SEQ ID NO: 4, and thePCR amplification and cloning protocol described above for cloning theLR33 myo-inositol 1-phosphate synthase from reverse transcribed mRNA. Inthis case, plasmid preparations from nine DH 10B clones that were shownto contain plasmid with the cDNA insert were pooled and used totransform electrocompetant DE 3 E. coli cells.

[0148] Kanamycin resistant colonies were selected and six were chosenfor inoculation of overnight cultures. The overnight cultures grown at30° in LB media with 30 mg 1⁻¹ kanamycin, were diluted two fold withfresh media, allowed to re-grow for 1 h, and induced by addingisopropyl-thiogalactoside to 1 mM final concentration.

[0149] Cells were harvested by centrifugation after 3 h and re-suspendedin 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mMphenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads wereadded and the mixture was sonicated three times for about five secondseach time with a microprobe sonicator. The mixture was centrifuged andthe protein concentration of the supernatant was determined. One μg ofprotein from the soluble fraction of each clonal culture was separatedby SDS-PAGE. Cultures which produced an addition protein band of about60 kilodaltons in mass were chosen for activity assay.

[0150] For the assay, [³³P]glucose-6-phosphate was prepared by thehexokinase catalyzed phosphorylation of glucose using [³³P]-γ-ATP as thephosphate donor. After 30 min at room temperature, the reaction mix waspassed through a SEP-PAC C18 column (MilliPore Corp. Milford, Mass.)which had been washed first with 80% methanol and then water. The columnpass through along with an additional 0.5 mL was collected and passedthrough a SEP-PAC SAX column which was then washed with 1 mL of 80%methanol. The [³³P]-glucose-6-phosphate was eluted with 2 mL of 0.02 NHCl in 80% methanol. Forty μl of 1 M Tris base was added and themethanol was removed under vacuum. The glucose-6-phosphate concentrationof the remaining solution was determined by its conversion to6-phospho-glucuronic acid by glucose-6-phosphate dehydrogenase. TheNADPH produced in the reaction was quantitated by absorbance at 340 nm.

[0151] Ten μL of the cell extracts were incubated at 37° for 30 min in100 μL reactions that were 2 mM in glucose-6-phosphate (57,334 dpm total³³P), 0.1 mM in NAD, and 15 mM in ammonium acetate. The reactions wereheated to 90° to precipitate protein, centrifuged to clear. Forty-sevenμL of the supernatant was applied to a Dionex™ PA-1 column run at 0.9 mlmin⁻¹ with 0.1 N NaOH and 0.1 N sodium acetate. Standards ofmyo-inositol 1-phosphate eluted from 8 through 10 min after injectionand 1 min fractions of the separated reaction mixes were taken throughthat time range for scintillation counting. The radioactivity in thepeak fractions was summed to obtain the conversion ofglucose-6-phosphate to myo-inositol-1-phosphate and the results for onecontrol E. coli DE 3 culture containing an empty pET24aT7 vector and twoclones containing the soybean myo-inositol 1-phosphate synthase cDNA areshown in Table 12. TABLE 12 The specific activity (μmoles myo-inositol1-phosphate produce min⁻¹ mg protein⁻¹) for three E. coli cell cultureextracts (Soy Clones 1 and 2 contain the soybean myo-inositol1-phosphate synthase while the control contains an empty plasmid.) LINESPECIFIC ACTIVITY CONTROL 0.024 μmol min⁻¹ mg⁻¹ SOY CLONE 1 0.524 μmolmin⁻¹ mg⁻¹ SOY CLONE 2 0.892 μmol min⁻¹ mg⁻¹

[0152] In the assay as run, both cDNA-containing clones used essentiallyall of the available substrate and were therefore more than 35 fold moreactive than the control line. The soybean myo-inositol 1-phosphatesynthase gene is therefore capable of producing a functional enzyme inE. coli.

[0153] The wild type soybean myo-inositol 1-phosphate synthase clonenumber 1 (Soy Clone 1) and three of the LR33 myo-inositol 1-phosphatesynthase clones were grown for protein expression as described above.Five μg of protein from the soluble cell extract from each clone wasseparated by SDS-PAGE. LR33 clone number 10 (LR33-10) produced asoluble, 60 kilodalton protein in essentially the same abundance as didthe wild type clone number 1. Four μg of protein from each of the twoextracts was assayed for myo-inositol 1-phosphate synthase activity bythe method described above using a 100 μL reaction that was 3 μm in NADand 90 μm in glucose-6-phosphate. The specific activity of the wild typemyo-inositol 1-phosphate synthase was 1.5 nmol min⁻¹ mg protein⁻¹ underthese conditions while the specific activity of the LR33-derivedmyo-inositol 1-phosphate synthase was 0.16 nmol min⁻¹ mg protein⁻¹.

[0154] The Nucleotide Sequence of the cDNA Encoding the LR33myo-inositol 1-phosphate Synthase

[0155] The nucleotide sequence of the cDNA insert in clone LR33-10(containing the nucleic acid fragment encoding the LR33 myo-inositol1-phosphate synthase) was determined using the DH 10 B E. coli strain ofthat clone as the plasmid source and DNA sequencing as described for thewild type clone. The nucleotide sequence is shown in SEQ ID NO: 5 andthe deduced amino acid sequence obtained from the open reading frame ofthat clone in SEQ ID NO: 6. SEQ ID NO: 5 differs by single base pairchange of G to T in the coding strand at base number 1241 from SEQ IDNO: 1. That change results in a change of amino acid number 396 fromlysine in the wild type sequence (SEQ ID NO: 2) to asparagine in theLR33 amino acid sequence (SEQ ID NO: 6).

[0156] To confirm that the base change resulted from a change in theLR33 genome rather than a PCR generated error which might have occurredduring the cloning of the LR33 myo-inositol 1-phosphate synthase, twosets of PCR primers were prepared. The wild type primer (SEQ ID NO: 7)and the LR33 primer (SEQ ID NO: 8) were used to amplify genomic DNAprepared from dry seeds of soybean cultivar A2872 and from soybean lineLR33 clone number 16 (see Table 8). The PCR primer described in SEQ IDNO: 4 was used as the common antisense strand primer. At annealingtemperatures of 62° or 64° and 35 cycles of annealing and extension,only the wild type primer produced a PCR product when A2872 DNA was usedas a template and only the primer corresponding to the LR33 sequenceproduced a product when LR33 DNA was used as template.

[0157] To further check the specificity of the primers for detecting themutation, DNA was prepared from six single plants grown from seed of thesegregating LR33 clone number 7 line described in Example 3 (see Table8). Out of six plants tested from the segregating population, two gaveDNA that acted as template for both primers, three gave DNA that actedas a primer only with the wild type primer, and one gave DNA thatproduced a product only with the LR33 primer. These are the resultsexpected from a population that contains heterozygotes containing onewild type and one mutant copy of the gene.

[0158] From the metabolite data and sequence data, we conclude that theobserved seed phenotype of very low total raffinose saccharide sugars,very high sucrose and low phytic acid are all due to the single basechange mutation described by the comparison of the wild type and LR33sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5.

Example 6 Transformation of Soybeans to Achieve Gene Silencing ofMyo-Inositol-1-Phosphate-Synthase and the Associated Seed Phenotype

[0159] Construction of Vectors for Transformation of Glycine max forReduced Expression of myo-inositol 1-phosphate Synthase in DevelopingSoybean Seeds

[0160] Plasmids containing the antisense or sense oriented soybeanmyo-inositol 1-phosphate synthase cDNA sequence under control of thesoybean Kunitz Trypsin Inhibitor 3 (KTi3) promoter [Jofuku and Goldberg,(1989) Plant Cell 1:1079-1093], the Phaseolus vulgaris 7S seed storageprotein (phaseolin) promoter [Sengupta-Gopalan et al., (1985) Proc.Natl. Acad. Sci. USA 82:3320-3324; Hoffman et al., (1988) Plant Mol.Biol. 11:717-729] and soybean β-conglycinin promoter [Beachy et al.,(1985) EMBO.J. 4:3047-3053], are constructed. The construction ofvectors expressing the soybean myo-inositol-1-phosphate synthase cDNAunder the control of these promoters is facilitated by the use of thefollowing plasmids: pML70, pCW108 and pCW109A.

[0161] The pML70 vector contains the KTi3 promoter and the KTi3 3′untranslated region and was derived from the commercially availablevector pTZ18R (Pharmacia) via the intermediate plasmids pML51, pML55,pML64 and pML65. A 2.4 kb Bst BI/Eco RI fragment of the complete soybeanKTi3 gene [Jofuku and Goldberg supra], which contains all 2039nucleotides of the 5′ untranslated region and 390 bases of the codingsequence of the KTi3 gene ending at the Eco RI site corresponding tobases 755 to 761 of the sequence described in Jofuku et al., (1989)Plant Cell 1:427-435, was ligated into the Acc I/Eco RI sites of pTZ18Rto create the plasmid pML51. The plasmid pML51 was cut with Nco I,filled in using Klenow, and religated, to destroy an Nco I site in themiddle of the 5′ untranslated region of the KTi3 insert, resulting inthe plasmid pML55. The plasmid pML55 was partially digested with XmnI/Eco RI to release a 0.42 kb fragment, corresponding to bases 732 to755 of the above cited sequence, which was discarded. A synthetic XmnI/Eco RI linker containing an Nco I site, was constructed by making adimer of complementary synthetic oligonucleotides consisting of thecoding sequence for an Xmn I site (5′-TCTTCC-3′) and an Nco I site(5′-CCATGGG-3′)followed directly by part of an Eco RI site(5′-GAAGG-3′). The Xmn I and Nco I/Eco RI sites were linked by a shortintervening sequence (5′-ATAGCCCCCCAA-3′). This synthetic linker wasligated into the Xmn I/Eco RI sites of the 4.94 kb fragment to createthe plasmid pML64. The 3′ untranslated region of the KTi3 gene wasamplified from the sequence described in Jofuku et al., [supra] bystandard PCR protocols (Perkin Elmer Cetus, GeneAmp PCR kit) using theprimers ML51 and ML52. Primer ML51 contained the 20 nucleotidescorresponding to bases 1072 to 1091 of the above cited sequence with theaddition of nucleotides corresponding to Eco RV (5-′GATATC-3′), Nco I(5′-CCATGG-3′), Xba I (5′-TCTAGA-3′), Sma I (5′-CCCGGG-3′) and Kpn I(5′-GGTACC-3′) sites at the 5′ end of the primer. Primer ML52 containedthe exact complement of the nucleotides corresponding to bases 1242 to1259 of the above cited sequence with the addition of nucleotidescorresponding to Sma I (5′-CCCGGG-3′), Eco RI (5′-GAATTC-3′), Bam HI(5′-GGATCC-3′) and Sal I (5′-GTCGAC-3′) sites at the 5′ end of theprimer. The PCR-amplified 3′ end of the KTi3 gene was ligated into theNco I/Eco RI sites of pML64 to create the plasmid pML65. A syntheticmultiple cloning site linker was constructed by making a dimer ofcomplementary synthetic oligonucleotides consisting of the codingsequence for Pst I (5′-CTGCA-3′), Sal I (5′-GTCGAC-3′), Bam HI(5′-GGATCC-3′) and Pst I (5′-CTGCA-3′) sites. The linker was ligatedinto the Pst I site (directly 5′ to the KTi3 promoter region) of pML65to create the plasmid pML70.

[0162] The pCW108 vector contains the bean phaseolin promoter and 3′untranslated region and was derived from the commercially availablepUC18 plasmid (Gibco-BRL) via plasmids AS3 and pCW104. Plasmid AS3contains 495 base pairs of the bean (Phaseolus vulgaris) phaseolin (7Sseed storage protein) promoter starting with 5′-TGGTCTTTTGGT-3′ followedby the entire 1175 base pairs of the 3′ untranslated region of the samegene [see sequence descriptions in Doyle et al., (1986)J. Biol. Chem.261:9228-9238 and Slightom et al., (1983) Proc. Natl. Acad. Sci. USA80:1897-1901; further sequence description may be found in World PatentPublication WO911/3993] cloned into the Hind III site of pUC18. Theadditional cloning sites of the pUC18 multiple cloning region (Eco RI,Sph I, Pst I and Sal I) were removed by digesting with Eco RI and Sal I,filling in the ends with Klenow and religating to yield the plasmidpCW104. A new multiple cloning site was created between the 495 bp ofthe 5′ phaseolin and the 1175 bp of the 3′ phaseolin by inserting adimer of complementary synthetic oligonucleotides consisting of thecoding sequence for a Nco I site (5′-CCATGG-3′) followed by three fillerbases (5′-TAG-3′), the coding sequence for a Sma I site (5′-CCCGGG-3′),the last three bases of a Kpn I site (5′-TAC-3′), a cytosine and thecoding sequence for an Xba I site (5′-TCTAGA-3′) to create the plasmidpCW108. This plasmid contains unique Nco I, Sma I, Kpn I and Xba I sitesdirectly behind the phaseolin promoter.

[0163] The vector pCW109A contains the soybean β-conglycinin promotersequence and the phaseolin 3′ untranslated region and is a modifiedversion of vector pCW109 which was derived from the commerciallyavailable plasmid pUC18 (Gibco-BRL). The vector pCW109 was made byinserting into the Hind III site of the cloning vector pUC18 a 555 bp 5′non-coding region (containing the promoter region) of the β-conglyciningene followed by the multiple cloning sequence containing therestriction endonuclease sites for Nco I, Sma I, Kpn I and Xba I, asdescribed for pCW108 above, then 1174 bp of the common bean phaseolin 3′untranslated region into the Hind III site (described above).

[0164] The β-conglycinin promoter region used is an allele of thepublished β-conglycinin gene [Doyle et al., (1986) J Biol. Chem.261:9228-9238] due to differences at 27 nucleotide positions. Furthersequence description of this gene may be found in World PatentPublication W091/13993.

[0165] These three nucleic acid constructions constitute seed specificexpression vectors with expression over a wide developmental periodincluding the period of myo-inositol synthesis for subsequent conversionto phytic acid. Insertion of the sequences described in SEQ ID NO: 1 andSEQ ID NO: 5 into these vectors is facilitated by the PCR methodsdescribed in Example 5 above. PCR primers which are complementary tochosen regions of SED ID NO: 1 or SEQ ID NO: 5 may be synthesized withadditional bases that constitute the recognition sequences ofrestriction endonucleases chosen from among those that also cut in themultiple cloning sequences following the promoter sequences of pML70,pCW108 and pCW109. Placement of the restriction sites may be chosen soas to direct the orientation of the nucleotide fragment from the soybeanmyo-inositol 1-phosphate synthase into the sense orientation to achieveeither over expression or co-suppression or into the antisenseorientation to achieve under expression.

[0166] Transformation of Somatic Soybean Embryo Cultures andRegeneration of Soybean Plants

[0167] The following stock solutions and media are used to support thegrowth of soybean tissues in vitro: Stock Solutions: MS Sulfate (100XStock) MS Halides (100X stock) (g/L) (g/L) MgSO₄ 7H₂O 37.0 CaCl₂ 2H₂O44.0 MnSO₄ H₂O 1.69 KI 0.083 ZnSO₄ 7H₂O 0.86 CoCl₂ 6H₂O 0.00125 CuSO₄5H₂O 0.0025 KH₂PO₄ 17.0 H₃BO₃ 0.63 Na₂MoO₄ 2H₂O 0.025 B5 Vitamin MSFeEDTA (100X stock) (g/L) (g/L) myo-inositol 10.0 Na₂EDTA 3.72 nicotinicacid 0.10 FeSO₄ 7H₂O 2.784 pyridoxine HCl 0.10

[0168] Media: Component (per Liter) SB55 SBP6 SB103 SB71-1 MS SulfateStock 10 mL 10 mL 10 mL — MS Halides Stock 10 mL 10 mL 10 mL — MS FeEDTAStock 10 mL 10 mL 10 mL — B5 Vitamin Stock 1 mL 1 mL — 1 mL 2,4-D Stock(10 mg/ml) 1 mL 0.5 mL — — Sucrose 60 g 60 g —      3% Maltose — —     6% — Asparagine 0.667 g 0.667 g — — NH₄NO 0.8 g 0.8 g — — KNO₃3.033 g 3.033 g — — MgCl₂ — — 750 mg 750 mg Gelrite — —    0.2%    0.2%pH 5.7 5.7 5.7 5.7

[0169] Soybean embryogenic suspension cultures are maintained in 35 mLliquid media (SB55 or SBP6) on a rotary shaker, 150 rpm, at 28° withmixed florescent and incandescent lights on a 16:8 h day/night schedule.Cultures are subcultured every four weeks by inoculating approximately35 mg of tissue into 35 mL of liquid medium.

[0170] Soybean embryogenic suspension cultures may be transformed withseed specific expression vectors containing either the sense orantisense oriented myo-inositol 1-phosphate synthase by the method ofparticle gun bombardment (see Kline et al. (1987) Nature (London)327:70). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) isused for these transformations.

[0171] To 50 mL of a 60 mg/mL 1 μm gold particle suspension are added(in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μlCaCl₂ (2.5 M). The particle preparation is agitated for 3 min, spun in amicrofuge for 10 sec and the supernatant removed. The DNA-coatedparticles are then washed once in 400 μL 70% ethanol and resuspended in40 μL of anhydrous ethanol. The DNA/particle suspension is sonicatedthree times for 1 sec each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

[0172] Approximately 300-400 mg of a four week old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. For each transformation experiment,approximately 5-10 plates of tissue were normally bombarded. Membranerupture pressure is set at 1000 psi and the chamber is evacuated to avacuum of 28 inches of mercury. The tissue is placed approximately 3.5inches away from the retaining screen and bombarded three times.Following bombardment, the tissue is placed back into liquid andcultured as described above.

[0173] Eleven days post bombardment, the liquid media is exchanged withfresh SB55 containing 50 mg/mL hygromycin. Thereafter, the selectivemedia is refreshed weekly. Seven weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Thus each new line is treated asindependent transformation event. These suspensions can then bemaintained as suspensions of embryos clustered in an immaturedevelopmental stage through subculture or regenerated into whole plantsby maturation and germination of individual somatic embryos.

[0174] Transformed embryogenic clusters are removed from liquid cultureand placed on a solid agar media (SB103) containing no hormones orantibiotics. Embryos are cultured for eight weeks at 26° with mixedflorescent and incandescent lights on a 16:8 h day/night schedule.During this period, individual embryos can be removed from the clustersand analyzed at various stages of embryo development. After eight weekssomatic embryos become suitable for germination. For germination, eightweek old embryos are removed from the maturation medium and dried inempty petri dishes for 1 to 5 days. The dried embryos are then plantedin SB71-1 medium were they are allowed to germinate under the samelighting and germination conditions described above. Germinated embryoscan then transferred to sterile soil and grown to maturity for seedcollection.

[0175] While in the globular embryo state in liquid culture as describedabove, somatic soybean embryos contain very low amounts oftriacylglycerol or storage proteins typical of maturing, zygotic soybeanembryos. At this developmental stage, the ratio of totaltriacylglyceride to total polar lipid (phospholipids and glycolipid) isabout 1:4, as is typical of zygotic soybean embryos at the developmentalstage from which the somatic embryo culture was initiated. At theglobular stage as well, the mRNAs for the prominent seed proteins(α′-subunit of β-conglycinin, Kunitz Trypsin Inhibitor 3 and SoybeanSeed Lectin) are essentially absent. Upon transfer to hormone-free mediato allow differentiation to the maturing somatic embryo state asdescribed above, triacylglycerol becomes the most abundant lipid class.As well, mRNAs for α′-subunit of β-conglycinin, Kunitz Trypsin Inhibitor3 and Soybean Seed Lectin become very abundant messages in the totalmRNA population. In these respects the somatic soybean embryo systembehaves very similarly to maturing zygotic soybean embryos in vivo.During the early maturation period the main soluble carbohydratespresent in the somatic embryos are sucrose, glucose and maltose (thesupplied sugar during the maturation phase). As the somatic embryosmature and begin to yellow, both raffinose and stachyose are formed. Inthis respect as well the phenotype of the somatic embryos is verysimilar to zygotic embryos as they go through the late stages of seeddevelopment. Thus selection for embryos that are transformed with seedspecific expression vectors which direct the expression of thenucleotide sequences described in SEQ ID NO: 1 or SEQ ID NO: 5 I ineither the sense or antisense orientation and that produce reducedamount of raffinose and stachyose should lead to the regeneration ofmature, fertile soybean plants which bear seeds with that samephenotype.

Example 7 Mutagenesis of Seeds and Identification of AdditionalPhosphorous Mutants

[0176] To obtain additional mutants with the instant phenotype, moresoybean seeds were mutagenized and screened.

[0177] Mutagenesis was accomplished by acquiring approximately 2,500soybean seeds (approximately 370 g) of twenty-one varieties. Seed wasimbibed in 2.5 liters of water for five hours with gentle aeration ofthe water. About 100 microliters of Antifoam-A® were added to reducefoaming. The seed was then drained in a colander and treated for threehours with 2.5 liters of 2.5 millimolar N-nitroso-N-methylurea (NMU) in0.1 molar sodium phosphate buffer with aeration as above.

[0178] After treatment, the NMU solution was poured into an appropriatewaste container and the seed was gently rinsed with water for ten tofifteen minutes. Following rinsing, the seeds were drained well, placedinto storage bags, and kept refrigerated until planting.

[0179] The mutagenized seed was gently planted by hand in shallowfurrows. Supplemental water was supplied via drip tubes to eliminate asmuch stress as possible from the emerging seedlings (“M0” plants). Mlseed was bulk harvested by mutagenized variety from the M0 plantstreated in this way.

[0180] A subset of the M1 seed was sent to winter nurseries and advancedtwo generations by a modified, single seed descent technique, well knownto those skilled in the art. A subset of harvested M3 seed was thenplanted. M3 plants are expected to be generally homozygous for mutationscaused earlier. Accordingly, M3 plants were harvested and threshedindividually, and M4 seed from each plant was identified and bulkedseparately. Several hundred thousand individual packets tracing tosingle plants were created in this way.

[0181] To qualitatively assay the inorganic phosphate content inindividual seeds, the seeds were placed in a multi-well tray, one seedper well. Using a press and a die machined especially for the tray, seedwas crushed to 2000 psi. This tray and press system was designed suchthat the tray accepted the crushing force of the hydraulic press, yetreturned to its original shape and retained added solvents withoutleaking. After pressure was released, residue on the crushing plate wasreturned to the tray. Crushed seed was then assayed by adding theacid/ammonium molybdate and reducing solution (described in Example 2above) directly to the seed residue in the wells.

[0182] The solution in wells containing seed expressing the high freephosphorus trait turned noticeably blue within ten to fifteen minutes ofcommencement of the assay, whereas seeds with normal phosphate levelsremained clear. All trays were scored soon after a thirty minuteincubation period at room temperature, because the samples in the wellswere observed to turn blue independent of the phosphorus concentration.

[0183] More than 60,000 mutagenized lines were examined in this mannerto identify 5 twenty-two potential phosphorus mutants. These results arerecorded in Table 13. TABLE 13 Genotypes Mutagenized, Number ofSelections Screened, and Resultant Putative Mutants Discovered PUTATIVEMUTANTS GENOTYPE SELECTIONS SCREENED* DISCOVERED** Variety 1 6059 1Variety 2 5666 0 Variety 3 2596 0 Variety 4 4396 6 Variety 5 2697 2Variety 6 2029 0 Variety 7 1210 0 Variety 8 3145 1 Variety 9 2343 0Variety 10 4623 5 Variety 11 3701 0 Variety 12 5611 4 Variety 13 1135 0Variety 14 4789 1 Variety 15 1076 0 Variety 16 3241 0 Variety 17 3275 0Variety 18 2505 2 TOTAL 60097  22 

[0184] Each plant that was identified as a potential phosphorus mutantwas tested again. Individual seeds of each line were submitted to thesame screening technique to confirm the original indication of elevatedfree phosphate. These results are reported in Table 14. TABLE 14Conformation of Original Positive Reading NUMBER OF INDIVIDUAL SEEDSTESTED POSTIVIE OF EACH PUTATIVE GENOTYPE REACTIONS MUTANT PutativeMutant 1 3 6 Putative Mutant 2 2 5 Putative Mutant 3 3 6 Putative Mutant4 25  25  Putative Mutant 5 3 6 Putative Mutant 6 3 6 Putative Mutant 74 6 Putative Mutant 8 2 6 Putative Mutant 9 5 6 Putative Mutant 10 3 6Putative Mutant 11 3 6 Putative Mutant 12 3 6 Putative Mutant 13 3 6Putative Mutant 14 5 6 Putative Mutant 15 5 6 Putative Mutant 16 6 6Putative Mutant 17 3 6 Putative Mutant 18 3 6 Putative Mutant 19 2 6Putative Mutant 20 1 6 Putative Mutant 21 6 6 Putative Mutant 22 6 6

[0185] To determine whether high non-phytate phosphorus mutants werealso expressing reduced levels of phytic acid, the levels ofmyo-inositol hexaphosphate were quantified. A 0.5 g sample portion ofground seed was placed in a 15 mL conical plastic centrifuge tube with 5mL 0.67 M HCl and homogenized for two minutes with a polytron tissuehomogenizer. The sample was extracted for 1 hour at room temperature,and was mixed once by vortexing. The extracted sample was placed in aclinical centrifuge at 2500 RPM for 15 minutes. A 2.5 mL volume ofsupernatant was removed and added to 25 mL water. This sample was thenapplied to a SAX® column (2 mL per minute). The column was washed with 1mL of 0.067 M HCl. The sample was then eluted from the column with 2 mLof 2 M HCl and evaporated to dryness at medium temperature on aSpeed-Vac. The dried sample was resuspended in 1 mL water and wasfiltered through a 0.45 micrometer syringe tip filter into a vial. A 10to 20 microliter sample was then prepared for injection into an HPLCcolumn.

[0186] The eluting solvent was prepared by mixing 515 mL of methanol,485 mL of double distilled water, 8 mL tetrabutyl ammonium hydroxide 40%(TBAH), 200 microliters of 10 N (5 M) sulfuric acid, 0.5 mL formic acidand 1-3 mg phytic acid. Phytic acid was prepared by placing 16 mg ofsodium phytate in 5 mL of water. This solution was placed on Dowex ionexchange resin (1 mL Dowex-50 acid form on glass wool in 5 mL pipettetip). This was rinsed with 1-2 mL water, and the filtrate brought to 10mL with water. The concentration is 1 mg/mL phytic acid. 2 mL is usedfor 1 liter of solvent. The pH of the solvent was adjusted to4.10+/−0.05 with 10 N sulfuric acid. Chromatography was accomplished bypumping the sample through a Hamilton PRP-1 reverse phase HPLC columnheated to 40° C. at a rate of 1 mL per minute. The detection of inositolphosphate is accomplished with a refractive index detector (Waters),which is auto-zeroed at least two minutes before each run.

[0187] Five confirmed phosphate mutants were tested in this manner. Fourof the mutants evaluated in this way were confirmed to be low inphytate. These results are reported in Table 15. Phytic acid reductionsof greater than sixty percent were found. TABLE 15 Confirmation ofPhytate Reduction GENOTYPE MUTAGENESIS WITH NMU PHYTATE* Wild type No(Control) 0.665 Wild type No (Control) 0.615 Wild type No (Control)0.565 Wild type No (Control) 0.525 Confirmed Mutant 5 Yes 0.385Confirmed Mutant 6 Yes 0.370 Confirmed Mutant 7 Yes 0.345 ConfirmedMutant 8 Yes 0.245 Confirmed Mutant 9 Yes 0.610

[0188] Of the five possible mutants tested for decreased phytic acidcontent, four were confirmed as positive. These four remaining mutantswere carried forward as breeding populations. Confirmed Mutant 5 (asreported in Table 15) was assigned the population number 29010CP01,Confirmed Mutant 7 was assigned 29004JP01, Confirmed Mutant 8 wasassigned 29018JP03 and Confirmed Mutant 6 was assigned 29018JP02. Insubsequent generations 29018JP02 proved to be unstable and wasdiscarded.

[0189] Analysis of soluble carbohydrates in populations 29004JP01 and29010CP01 also indicate the low total raffinose saccharide phenotypefound in mutant LR33. The soluble carbohydrate analysis on at least onepopulation of 29018JP03 appeared to be that of a segregating populationlike that first seen with LR33.

[0190] Thus, it has been shown by this example that the low phytic acid,increased inorganic phosphate, low raffinosaccharide phenotype isconsistently obtainable by the mutagenesis and screening methoddescribed herein.

Example 8 Nucleotide and Deduced Amino Acid Sequences of Myo-Inositol1-Phosphate Synthases from Additional Low Phtate Mutants

[0191] Developing seed from the three confirmed mutant populations(described in Example 7 above) was harvested during the first one-thirdof the pod filling period and mRNA was extracted and purified as inExample 5. First strand cDNA was prepared again as in Example 5, and thePCR primers shown in SEQ ID NO: 3 and SEQ ID NO: 5 were used to amplifymyo-inositol 1-phosphate synthase cDNAs from each of the three mutants.The cDNA inserts that were thus obtained were cloned into the pET24avector as described in Example 5, used to transform E. coli DH10α cellsand the plasmid DNA obtained from these cells was sequenced. SEQ ID NO:9 sets forth the sequence of cDNA from 29004JP01, and the correspondingdeduced amino acid sequence for the myo-inositol 1-phosphate synthase ispresented as SEQ ID NO: 10. The cDNA sequence for 29010CP01 is shown asSEQ ID NO: 11, with the corresponding deduced amino acid sequence ofmyo-inositol 1-phosphate synthase in SEQ ID NO: 12. The sequence for29018JP03 is shown in SEQ ID NO: 13, and the corresponding deduced aminoacid sequence for that mutant myo-inositol 1-phosphate synthase as SEQID NO: 14.

[0192] Sequence comparisons with the wild type and mutant (LR33)myo-inositol 1-phosphate synthase sequences described in Example 5indicated that while the sequence of the cDNA obtained from 29004CP01 isidentical to the sequence described as the wild type sequence in Example5 (hereinafter the “wt1 allele”; designated SEQ ID NO: 1 in FIG. 2), thenucleotide sequences of the cDNAs from 29010CP01 and 29018JP03 eachdiffer at 42 positions when compared to wtl allele (see FIG. 2).

[0193] In an effort to explain these differences, the sequence of anadditional wild type cDNA was obtained from an EST collection derivedfrom a cDNA library made from mRNA from developing seeds of the soybeancultivar Wye. The coding region of the full insert in EST clone S2.15e07is shown in SEQ ID NO: 15; the deduced amino acid sequence for theencoded product is set forth in SEQ ID NO: 16. The DNA sequence of thecoding region of S2.15e07, hereinafter referred to as the “wt2 allele”,is shown as SEQ ID NO: 15 in FIG. 2. When the sequence of 29018JP03 iscompared to that of wt2 allele, no differences are detectable. Thesequence of the cDNA from 29010CP01 (SEQ ID NO: 11, see FIG. 2) has asingle base change located at base 260 when compared to all the othersequences. Since seeds of this line (i.e., Wye) are normal with respectof inorganic phosphate and phytic acid levels, and since myo-inositol1-phosphate synthase enzyme encoded by the nucleotide sequence of thewt2 allele is of normal specific activity, we conclude that there ismore than one wild type allele in soybean.

[0194] A comparison of the deduced amino acid sequences for all sixsequences is shown in FIG. 3. The two wild type alleles (SEQ ID Nos: 2and 16) differ by seven residues; both LR33 and 29010CP01 differ by anadditional amino acid change in comparison to sequences of the the wildtype alleles that they most resemble (wt1 and wt2, respectively). Whenexpressed in E. coli, 29010CP01 produced an enzyme with low specificactivity, whereas 29018JP03 produced an enzyme with activity comparableto that encoded by the wt1 allele. These enzymes were purified andassayed as in Example 5.

[0195] We conclude that both wild type sequences encode an activemyo-inositol 1-phosphate synthase, and that single amino acid changes ineither of the isoforms can lead to decreased enzyme activity.

Example 9 Genetic Tests of Allelism among the Mutant Lines

[0196] Neither 29004JP01 nor 29018JP03 have changes in the coding regionof their cDNAs for myo-inositol 1-phosphate synthase when compared toknown wild type sequences. This could be explained if the message levelfor myo-inositol 1-phosphate synthase is decreased in these lines. It isnot clear from the data if the sequences of the wt1 and wt2 alleles aremembers of a gene family within soybean or if they represent allelicvariants within the soybean genome.

[0197] To determine if all four mutations reside at the same geneticlocus, crosses were made between a homozygous descendant of LR33 and of29004JP01 and between LR33 and 29018JP03. The F1 seeds from both crosseswere harvested at maturity, a chip of cotyledon that was oriented awayfrom the embryonic axis was remove and tested for free phosphate in thequalitative assay described in Examples 4 and 7. All seeds tested highin inorganic phosphate in comparison to chips from wild typeseeds. Sincethe mutations in LR33, 29004JP01 and 29018JP03 are all recessive, thehigh phosphate phenotype should not be present in the F1 seed unless allthree mutations are at the same locus. After analysis, the remainder ofeach F1 seed was planted and the F1 plants were allowed to selfpollinate to produce the F1:2 seed. Twelve seeds from one plant fromeach cross were ground individually and inorganic phosphate wasdetermined quantitatively as described in Example 4. Twelve seeds from awild type control and from an LR33 homozygote grown in the sameenvironment were analyzed as controls. The results are shown in Table16. TABLE 16 The inorganic phosphate content of single seeds form thesegregating F2 seed population from crosses between LR33 and twoindependent low phytic acid mutants. Seed 29010CP01 Wild typeLR33x29004JP01 LR33x29018JP03 Number (Micromoles Inorganic Phosphate perGram Seed Weight) 1 1.369 0.194 1.176 0.895 2 1.342 0.190 1.031 1.047 31.750 0.182 1.008 0.768 4 1.752 0.204 0.779 0.924 5 1.117 0.199 0.9240.990 6 1.102 0.137 0.925 0.919 7 1.336 0.187 0.867 0.891 8 1.392 0.1280.977 0.964 9 0.991 0.181 0.830 1.034 10 1.560 0.137 0.538 1.278 111.147 0.146 0.999 1.054 12 1.240 0.151 1.230 1.226

[0198] All twelve seeds from both of the F2 seed populations have thehigh inorganic phosphate phenotype, which is comparable to thehomozygous control and is much higher the wild type seeds. Since thereis also no segregation for phenotype in the F2 seed population, weconclude that all three of the mutant lines tested carry genetic defectsat the genetic locus. The locus defined by the tests described in thisExample is designated Mips1.

[0199] The sequences of the wt1 and wt2 alleles apparently represent twoallelic variants present in the domesticated soybean gene pool. It isnot expected that both wild type alleles would be present in onecultivar. In order to better understand the basis for this unexpectedobservation, a second line of evidence was studied. In PCR amplificationof the myo-inositol 1-phosphate synthase gene from several commercialsoybean cultivars, a difference in intron length was noted betweencultivars. Cultivars from Asgrow Seed Co. designated A2271, a2704,A2850, A3160 and A3304 all contain a 340 base pair intron between aminoacids 471 and 472 of sequences shown in FIG. 3, as do the mutant linesLR33 and 29004JP01. Asgrow cultivars A3002 and A3244 both contain a 300base pair intron between these same amino acids, as do mutant lines29018JP03 and 29010CP01. Further, the same PCR amplification performedon DNA from the F1 plants resulting from the crosses of LR33 and of29004JP01 and between LR33 and 29018JP03 described above reveal bothintron lengths. The combinations of these results strongly suggest thatboth wild type sequences are in fact alleles of the same locus, thatboth do not exist in one homozygous plant and therefore that thecultivar Wye from which both wild type sequences were obtained is eitherimpure or segregating at that locus.

[0200] Mutations that cause either a decrease in the activity of enzymesencoded by these sequences or in the amount of message or proteinproduced by either of these genes are capable of giving the combined lowphytic acid, high inorganic phosphate, low raffinosaccharide phenotypedescribed in Example 4.

What is claimed is:
 1. An isolated nucleic acid fragment encoding asoybean myo-inositol 1-phosphate synthase.
 2. The nucleic acid fragmentof claim 1 wherein the nucleotide sequence encoding the soybeanmyo-inositol 1-phosphate synthase is substantially similar to thenucleotide sequence set forth in a member selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO:
 15. 3. The nucleic acidfragment of claim 1 wherein the nucleotide sequence encoding the soybeanmyo-inositol 1-phosphate synthase encodes the amino acid sequence setforth in a member selected from the group consisting SEQ ID NO: 2 andSEQ ID NO:
 16. 4. The nucleic acid fragment of claim 1 wherein thenucleotide sequence encoding the soybean myo-inositol 1-phosphatesynthase is set forth in a member selected from the group consisting SEQID NO: 1 and SEQ ID NO:
 15. 5. A chimeric gene comprising the nucleicacid fragment of claim 1 or the complement of the nucleic acid fragmentof claim 1, operably linked to suitable regulatory sequences.
 6. Achimeric gene comprising a subfragment of the nucleic acid fragment ofclaim 1 or the complement of a subfragment of the nucleic acid fragmentof claim 1, operably linked to suitable regulatory sequences, whereinexpression of the chimeric gene results in a decrease in expression ofan endogenous or native gene encoding a soybean myo-inositol 1-phosphatesynthase.
 7. An isolated nucleic acid fragment encoding a mutantmyo-inositol 1-phosphate synthase having decreased capacity for thesynthesis of myo-inositol-1-phospate.
 8. The nucleic acid fragment ofclaim 7 wherein the nucleotide sequence encoding the nutant myo-inositol1-phosphate synthase is substantially similar to the nucleotide sequenceset forth in a member selected from the group consisting SEQ ID NO: 5and SEQ ID NO:
 11. 9. The nucleic acid fragment of claim 7 wherein thenucleotide sequence encoding the mutant myo-inositol 1-phosphatesynthase encodes the amino acid sequence set forth in a member selectedfrom the group consisting SEQ ID NO: 6 and SEQ ID NO:
 12. 10. Thenucleic acid fragment of claim 7 wherein the nucleotide sequenceencoding the mutant myo-inositol 1-phosphate synthase is set forth in amember selected from the group consisting SEQ ID NO: 5 and SEQ ID NO:11.
 11. A soybean plant with a heritable phenotype of (i) a seed phyticacid content of less than 17 μmol/g, (ii) a seed content of raffinoseplus stachyose of less than 14.5 μmol/g, and (iii) a seed sucrosecontent of greater than 200 μmol/g, provided that the plant is not LR33.12. The soybean plant of claim 11 wherein the soybean plant ishomozygous for a genetic defect at the Mips 1 locus.
 13. The soybeanplant of claim 12 wherein the soybean plant bears ATCC Accession No.97971.
 14. The soybean plant of claim 12 wherein the soybean plant bearsATCC Accession No. XXXXX.
 15. The soybean plant of claim 12 wherein thesoybean plant bears ATCC Accession No. YYYYY.
 16. The soybean plant ofclaim 12 wherein the soybean plant bears ATCC Accession No. ZZZZZ. 17.The soybean plant of claim 11 wherein the soybean plant is homozygousfor at least one gene encoding a mutant myo-inositol 1-phosphatesynthase having decreased capacity for the synthesis of myo-nositol1-phosphate.
 18. The soybean plant of claim 17 comprising the nucleicacid fragment of claim
 7. 19. Seeds of the soybean plant of claim 11.20. A soybean plant comprising the chimeric gene of claim 5 or claim 6wherein the soybean plant has a heritable phenotype of (i) a seed phyticacid content less than 17 μmol/g, (ii) a seed content of raffinose plusstachyose of less than 14.5 μmol/g, and (iii) a seed sucrose content ofgreater than 200 μmol/g.
 21. Seeds of the soybean plants of claim 20.22. A method for making a soybean plant with a heritable phenotype of(i) a seed phytic acid content less than 17 μmol/g, (ii) a seed contentof raffinose plus stachyose of less than 14.5 μmol/g, and (iii) a seedsucrose content of greater than 200 μmol/g, the method comprising: (a)crossing LR33 or the soybean plant of claim 11 with an elite soybeanplant; and (b) selecting a progeny plant of the cross of step (a) thathas a heritable phenotype of (i) a seed phytic acid content less than 17μmol/g, (ii) a seed content of raffinose plus stachyose of less than14.5 μmol/g, and (iii) a seed sucrose content of greater than 200μmol/g.
 23. Seeds of the soybean plant made by the method of claim 22.24. A method for making a soybean plant with a heritable phenotype of(i) a seed phytic acid content less than 17 μmol/g, (ii) a seed contentof raffinose plus stachyose of less than 14.5 μmol/g, and(iii) a seedsucrose content of greater than 200 μmol/g, the method comprising: (a)crossing the soybean plant of claim 20 with an elite soybean plant; and(b) selecting progeny plant of the cross of step (a) that has aheritable phenotype of (i) a seed phytic acid content less than 17μmol/g, (ii) a seed content of raffinose plus stachyose of less than14.5 μmol/g, and (iii) a seed sucrose content of greater than 200μmol/g.
 25. Seeds of the soybean plant made by the method of claim 24.26. A soy protein product derived from seeds of a soybean planthomozygous for at least one gene encoding a mutant myo-inositol1-phosphate synthase having decreased capacity for the synthesis ofmyo-inositol 1-phosphate, the gene conferring a heritable phenotype of(i) a seed phytic acid content less than 17 μmol/g, (ii) a seed contentof raffinose plus stachyose of less than 14.5 μmol/g, and (iii) a seedsucrose content of greater than 200 μmol/g.
 27. A soy protein productderived from the processing of soybean seeds of claim
 19. 28. A soyprotein product derived from the processing of soybean seeds of claim21.
 29. A soy protein product derived from the processing of soybeanseeds of claim
 23. 30. A soy protein product derived from the processingof soybean seeds of claim
 25. 31. A method for making a soy proteinproduct derived from seeds of a soybean plant with a heritable phenotypeof (i) a seed phytic acid content less than 17 μmol/g, (ii) a seedcontent of raffinose plus stachyose of less than 14.5 μmol/g, and (iii)a seed sucrose content of greater than 200 μmol/g comprising: (a)crossing an agronomically elite soybean plant with LR33 or the soybeanplant of claim 11; (b) screening the seed of progeny plants obtainedfrom step (a) for (i) a seed phytic acid content less than 17 μmol/g,(ii) a seed content of raffinose plus stachyose of less than 14.5μmol/g, and (iii) a seed sucrose content of greater than 200 μmol/g; and(c) processing the seed selected in step (b) to obtain the desiredsoybean protein product.
 32. A method for producing a soy proteinproduct derived from seeds of a soybean plant with a heritable phenotypeof (i) a seed phytic acid content less than 17 μmol/g, (ii) a seedcontent of raffinose plus stachyose of less than 14.5 μmol/g, and (iii)a seed sucrose content of greater than 200 μmol/g comprising: (a)crossing an agronomically elite soybean plant with the soybean plant ofclaim 20; (b) screening the seed of progeny plants obtained from step(a) for (i) a seed phytic acid content less than 17 μmol/g, (ii) a seedcontent of raffinose plus stachyose of less than 14.5 μmol/g, and (iii)a seed sucrose content of greater than 200 μmol/g; and (c) processingthe seed selected in step (b) to obtain the desired soybean proteinproduct.
 33. A method of using a soybean plant homozygous for at leastone gene encoding a mutant myo-inositol 1-phosphate synthase havingdecreased capacity for the synthesis of myo-inositol 1-phosphate, thegene conferring a heritable phenotype of (i) a seed phytic acid contentless than 17 μmol/g, (ii) a seed content of raffinose plus stachyose ofless than 14.5 μmol/g, and (iii) a seed sucrose content of greater than200 μmol/g to produce progeny lines, the method comprising: (a) crossinga soybean plant comprising a mutant myo-inositol 1-phosphate synthasehaving decreased capacity for the synthesis of myo-inositol 1-phosphatewith any soybean parent which does not comprise the mutation, to yield aF1 hybrid; (b) selfing the F1 hybrid for at least one generation; and(c) identifying the progeny of step (b) homozygous for at least one geneencoding a mutant myo-inositol 1-phosphate synthase having decreasedcapacity for the synthesis of myo-inositol 1-phosphate, the geneconferring a heritable phenotype of (i) a seed phytic acid content lessthan 17 μmol/g, (ii) a seed content of raffinose plus stachyose of lessthan 14.5 μmol/g, and (iii) a seed sucrose content of greater than 200μmol/g.